Compositions for drug administration

ABSTRACT

A composition having at least one alkyl glycoside and at least one therapeutic agent, wherein the alkylglycoside has an alkyl chain length from about 10 to about 16 carbon atoms and wherein the therapeutic agent is an oligonucleotide.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation-in-part and claims the benefit ofpriority under 35 U.S.C. §120 of U.S. application Ser. No. 11/193,825,filed Jul. 29, 2005, currently pending, which is a continuation-in-partand claims the benefit of priority under 35 U.S.C. §120 of U.S.application Ser. No. 11/127,786, filed May 11, 2005, currently pending,and claims the benefit of priority under 35 U.S.C. §119(e) of U.S.Application Ser. No. 60/649,958, filed Feb. 3, 2005; the benefit ofpriority under 35 USC §119(e) of U.S. Application Ser. No. 60/637,284,filed Dec. 17, 2004; the benefit of priority under 35 USC §119(e) ofU.S. Application Ser. No. 60/632,038, filed Nov. 30, 2004; the benefitof priority under 35 USC §119(e) of U.S. Application Ser. No.60/609,890, filed Sep. 14, 2004; and the benefit of priority under 35USC §119(e) of U.S. Application Ser. No. 60/604,296, filed Aug. 25,2004. The disclosure of each of the prior applications is consideredpart of and is incorporated by reference in the disclosure of thisapplication.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to non-irritating, non-toxiccompositions providing enhanced bioavailability and more specifically toalkyl glycoside or saccharide alkyl ester compositions for delivery oftherapeutic agents to a subject.

2. Background Information

Therapeutic agents are often combined with various surfactants. Yet,surfactants are frequently irritating to the skin and other tissues,including mucosal membranes such as those found in the nose, mouth, eye,vagina, rectum, esophagus, intestinal tract, and the like. Manysurfactants also cause proteins to denature, thus destroying theirbiological activity. Another serious limitation to the development anduse of such agents is the ability to deliver them safely,non-invasively, efficiently and stably to the site of action. Therefore,an ideal enhancing surfactant will stabilize the therapeutic agent, benon-toxic and non-irritable to the skin or mucosal surfaces, haveantibacterial activity, and enhance the passage or absorption of thetherapeutic agent through various membrane barriers without damaging thestructural integrity and biological function of the membrane andincrease bioavailability of the agent.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the development of atherapeutic composition containing a drug enhancing agent useful forincreasing the absorption and bioavailability of the drug, while at thesame time avoiding various adverse toxic effects of drug. In particular,the drug enhancing agents of the invention contain a non-toxicsurfactant consisting of at least an alkyl glycoside and/or saccharidealkyl ester. One advantage of the therapeutic compositions of theinvention is that they permit administration and delivery of thetherapeutic agents with high bioavailabilities at concentrations ofenhancing agents that are dramatically below their so-called “noobservable adverse effect levels” (their NOAEL's). Accordingly, thepresent invention provides compositions, including alkyl glycosidesand/or saccharide alkyl esters and a therapeutic agent (e.g. smallmolecule organic drug molecules, low molecular weight peptides such asExenatide, GLP-1 and the like, proteins, and non-peptide therapeuticpolymers such as low molecular weight heparin and inhibitory RNA),methods of administering and using the compositions e.g. via the oral,ocular, nasal, nasolacrimal, inhalation or pulmonary, oral cavity(sublingual or Buccal cell) or cerebral spinal fluid (CSF) deliveryroute, and methods of ameliorating a disease state in a subject byadministration of such compositions

In one aspect, the present invention relates to a surfactant compositionhaving at least one alkyl glycoside and/or at least one saccharide alkylester, and when admixed, mixed or blended with a therapeutic agent, adrug, or biologically active compound, the surfactant stabilizes thebiological activity and increases the bioavailability of the drug.

Accordingly, in one aspect, the invention provides a therapeuticcomposition having at least one biologically active compound and atleast one surfactant, wherein the surfactant further consists of atleast one alkyl glycoside and/or saccharide alkyl ester or sucrose esterand wherein the therapeutic composition stabilizes the biologicallyactive compound for at least about 6 months, or more, and from about 4°C. to about 25° C.

The invention also provides a method of administering a therapeuticcomposition having a surfactant including at least one alkyl glycosideand/or saccharide alkyl ester admixed, mixed, or blended with at leastone therapeutic agent, or a drug, or biologically active compound, andadministered or delivered to a subject, wherein the alkyl has from about10 to 24, 10 to 20, 10 to 16, or 10 to 14 carbon atoms, wherein thesurfactant increases the stability and bioavailability of thetherapeutic agent.

In yet another aspect, the invention provides a method of increasingabsorption of a low molecular weight compound into the circulatorysystem of a subject by administering the compound via the oral, ocular,nasal, nasolacrimal, inhalation or pulmonary, oral cavity (sublingual orBuccal cell), or CSF delivery route when admixed, mixed or blended withan absorption increasing amount of a suitable surfactant, wherein thesurfactant is a nontoxic and nonionic hydrophobic alkyl joined by alinkage to a hydrophilic saccharide. Such low molecular weight compoundsinclude but are not limited to, nicotine, interferon, PYY, GLP-1,synthetic exendin-4, parathyroid hormone, human growth hormone, or asmall organic molecule. Additional low molecular weight compoundsinclude antisense oligonucleotides or interfering RNA molecules (e.g.,siRNA or RNAi).

The present invention also provides a method of treating diabetesincluding administering to a subject in need thereof via the oral,ocular, nasal, nasolacrimal, inhalation or pulmonary, or oral cavity(sublingual or Buccal cell), a blood glucose reducing amount of atherapeutic composition, for example, an incretin mimetic agent or afunctional equivalent thereof, and an absorption increasing amount of asuitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkylgroup joined by a linkage to a hydrophilic saccharide, therebyincreasing the absorption of incretin mimetic agent or insulin andlowering the level of blood glucose and treating diabetes in thesubject.

The present invention also provides a method of treating congestiveheart failure in a subject including administering to the subject inneed thereof via the oral, ocular, nasal, nasolacrimal, or inhalationdelivery route, a therapeutically effective amount of a compositioncomprising a GLP-1 peptide or a functional equivalent thereof, and anabsorption increasing amount of a suitable nontoxic, nonionic alkylglycoside having a hydrophobic alkyl joined by a linkage to ahydrophilic saccharide, thereby treating the subject.

In another aspect, the invention provides a method of treating obesityor diabetes associated with obesity in a subject comprisingadministering to a subject in need thereof via the oral, ocular, nasal,nasolacrimal, inhalation or CSF delivery route, a therapeuticallyeffective amount of a composition comprising a PYY peptide or afunctional equivalent thereof, and an absorption increasing amount of asuitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkyljoined by a linkage to a hydrophilic saccharide, thereby treating thesubject.

In another aspect, the invention provides a method of increasingabsorption of a low molecular weight therapeutic compound into thecirculatory system of a subject by administering via the oral, ocular,nasal, nasolacrimal, inhalation or CSF delivery route the compound andan absorption increasing amount of a suitable nontoxic, nonionic alkylglycoside having a hydrophobic alkyl group joined by a linkage to ahydrophilic saccharide, wherein the compound is from about 1-30 kD, withthe proviso that the compound is not insulin, calcitonin, or glucagonwhen the route of administration is oral, ocular, nasal, ornasolacrimal.

The present invention also provides a method of increasing absorption ofa low molecular weight therapeutic compound into the circulatory systemof a subject by administering via the oral, ocular, nasal, nasolacrimal,inhalation or pulmonary, oral cavity (sublingual or Buccal cell) or CSFdelivery route the compound and an absorption increasing amount of asuitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkyljoined by a linkage to a hydrophilic saccharide, wherein the compound isfrom about 1-30 kilo Daltons (kD), with the proviso that the subjectdoes not have diabetes when delivery is via the oral, ocular, nasal ornasolacrimal routes.

In one aspect of the invention, there is provided a pharmaceuticalcomposition having a suitable nontoxic, nonionic alkyl glycoside havinga hydrophobic alkyl group joined by a linkage to a hydrophilicsaccharide in combination with a therapeutically effective amount ofExenatide (exendin-4) in a pharmaceutically acceptable carrier.

In one aspect, the invention provides a pharmaceutical compositionhaving a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount of GLP-1 in apharmaceutically acceptable carrier.

In one aspect, the invention provides a pharmaceutical compositionhaving a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount of nicotine in apharmaceutically acceptable carrier.

In one aspect, the invention provides a pharmaceutical compositioncomprising a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount of interferon ina pharmaceutically acceptable carrier.

In one aspect, the invention provides pharmaceutical composition havinga suitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkylgroup joined by a linkage to a hydrophilic saccharide in combinationwith a therapeutically effective amount of PYY in a pharmaceuticallyacceptable carrier.

In one aspect, the invention provides a pharmaceutical compositionhaving a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount of parathyroidhormone in a pharmaceutically acceptable carrier.

In one aspect, the invention provides a pharmaceutical compositionhaving a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount of a peptidehaving a molecular weight of about 1-75 kD in a pharmaceuticallyacceptable carrier, with the proviso that the peptide is not insulin,calcitonin, and glucagon.

In one aspect, the invention provides a pharmaceutical compositionhaving a suitable nontoxic, nonionic alkyl glycoside having ahydrophobic alkyl group joined by a linkage to a hydrophilic saccharidein combination with a therapeutically effective amount erythropoietin ina pharmaceutically acceptable carrier.

In one aspect, the invention provides a pharmaceutical compositionhaving a therapeutically effective amount of an oligonucleotide incombination with an absorption increasing amount of an alkylglycoside.The oligonucleotide can be an antisense oligonucleotide or interferingRNA molecules, such as siRNA or RNAi. The oligonucleotide typically hasa molecular weight of about 1-20 kD and is from about 1-100, 1-50, 1-30,1-25 or 15-25 nucleotides in length. In another aspect, theoligonucleotide has a molecular weight of about 5-10 kD. In one aspect,the alkylglycoside is tetradecyl-beta-D-maltoside.

In yet another aspect, the invention provides a method of increasing thebioavailability of a low molecular weight oligonucleotide in a subjectby administering the compound with an absorption increasing amount of analkylglycoside, thereby increasing the bioavailability of the compoundin the subject. In one aspect, the alkylglycoside istetradecyl-beta-D-maltoside.

In one aspect, the invention provides a method of increasing absorptionof a compound into the CSF of a subject having administered intranasallythe compound and an absorption increasing amount of a suitable nontoxic,nonionic alkyl glycoside having a hydrophobic alkyl group joined by alinkage to a hydrophilic saccharide.

In yet another aspect, the invention provides a pharmaceuticalcomposition having a suitable nontoxic, nonionic alkyl glycoside havinga hydrophobic alkyl group joined by a linkage to a hydrophilicsaccharide in combination with a mucosal delivery-enhancing agentselected from:

(a) an aggregation inhibitory agent;

(b) a charge-modifying agent;

(c) a pH control agent;

(d) a degradative enzyme inhibitory agent;

(e) a mucolytic or mucus clearing agent;

(f) a ciliostatic agent;

(g) a membrane penetration-enhancing agent selected from:

-   -   (i) a surfactant; (ii) a bile salt; (ii) a phospholipid        additive, mixed micelle, liposome, or carrier; (iii) an        alcohol; (iv) an enamine; (v) an NO donor compound; (vi) a        long-chain amphipathic molecule; (vii) a small hydrophobic        penetration enhancer; (viii) sodium or a salicylic acid        derivative; (ix) a glycerol ester of acetoacetic acid; (x) a        cyclodextrin or beta-cyclodextrin derivative; (xi) a        medium-chain fatty acid; (xii) a chelating agent; (xiii) an        amino acid or salt thereof; (xiv) an N-acetylamino acid or salt        thereof; (xv) an enzyme degradative to a selected membrane        component; (ix) an inhibitor of fatty acid synthesis; (x) an        inhibitor of cholesterol synthesis; and (xi) any combination of        the membrane penetration enhancing agents recited in (i)-(x);

(h) a modulatory agent of epithelial junction physiology;

(i) a vasodilator agent;

(j) a selective transport-enhancing agent; and

(k) a stabilizing delivery vehicle, carrier, mucoadhesive, support orcomplex-forming species with which the compound is effectively combined,associated, contained, encapsulated or bound resulting in stabilizationof the compound for enhanced nasal mucosal delivery, wherein theformulation of the compound with the intranasal delivery-enhancingagents provides for increased bioavailability of the compound in a bloodplasma of a subject.

In one aspect, the invention provides a method of increasing absorptionof a low molecular weight compound into the circulatory system of asubject by administering, via the oral, ocular, nasal, nasolacrimal,inhalation or pulmonary, oral cavity (sublingual or Buccal cell) or CSFdelivery route (a) the compound; (b) an absorption increasing amount ofa suitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkylgroup joined by a linkage to a hydrophilic saccharide; and (c) a mucosaldelivery-enhancing agent.

In one aspect, the invention provides a method of controlling caloricintake by administering a composition having a therapeutic effectiveamount of exendin-4, or related GLP-1 peptide, with an effective amountof Intravail alkyl saccharide.

In another aspect, the invention provides a method of controlling bloodglucose levels in a subject by administering to a subject a compositioncomprising a therapeutic effective amount of exendin-4, or related GLP-1peptide, with an effective amount of Intravail alkyl saccharide.

Still, in another aspect, the invention provides a controlled releasedosage composition comprising:

-   -   (a) a core comprising:        -   (i) at least one therapeutic agent or drug;        -   (ii) at least one alkyl glycoside and/or saccharide alkyl            ester; and    -   (b) at least one membrane coating surrounding the core, wherein        the coating is impermeable, permeable, semi-permeable or porous        and becomes more permeable upon sustained contact with contents        of the gastrointestinal tract.

In another embodiment, the invention provides a method of administeringan alkylglycoside composition by administering a therapeuticallyeffective amount of at least one alkylglycoside having an alkyl chainlength from about 12 to about 14 carbon atoms, at least one saccharidewith an antibacterial activity, and at least one therapeutic agent.

Still in another embodiment, the invention provides a composition havingat least one drug selected from the group consisting of insulin, PYY,Exendin-4 or other GLP-1 related peptide, human growth hormone,calcitonin, parathyroid hormone, truncated parathyroid hormone peptidessuch as PTH 1-34, EPO, interferon alpha, interferon beta, interferongamma, and GCSF and at least one alkyl saccharide having antibacterialactivity.

In one aspect, the invention provides an antibacterial alkyl saccharidecomposition, which includesn-Dodecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside orn-tetradecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside.

Yet, in another aspect, the invention provides an aqueous drugcomposition for transmucocal or transdermal administration having atleast one drug and at least one antibacterial agent in a concentrationfrom about 0.05% to about 0.5%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the intranasal percent bioavailabilitycompared to intravenous injection and the subject-to-subjectcoefficients of variation for MIACALCIN® (salmon calcitonin) with andwithout alkyl glycoside.

FIG. 2 is a graph showing the effect of intranasal administration ofinsulin/0.25% TDM (filled circles) and intranasal administration ofinsulin alone (open circles) in reducing blood glucose levels.

FIG. 3 is a graph showing the effect of intranasal (closed triangles)and intraperitoneal (IP) injection (closed circles) administration ofexendin-4/0.25% TDM and IP injection of saline alone, minus TDM (opencircles) in reducing blood glucose levels following intraperitoneal (IP)injection of glucose (i.e., in a so-called “glucose tolerance test”).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesincluded therein.

The present invention is based on the discovery that therapeuticcompositions comprising of least one drug and at least one surfactant,wherein the surfactant is comprised of at least one alkyl glycosideand/or at least one saccharide alkyl ester are stable, non-toxic,non-irritating, anti-bacterial compositions that increasebioavailability of the drug and have no observable adverse effects whenadministered to a subject.

A “therapeutic composition” can consist of an admixture with an organicor inorganic carrier or excipient, and can be compounded, for example,with the usual non-toxic, pharmaceutically acceptable carriers fortablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, or other form suitable for use. The carriers, in additionto those disclosed above, can include glucose, lactose, mannose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, mediumchain length triglycerides, dextrans, and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form.In addition, auxiliary stabilizing, thickening or coloring agents can beused, for example a stabilizing dry agent such as triulose.

A “drug” is any therapeutic compound, or molecule, or therapeutic agent,or biologically active compound, including but not limited to nucleicacids, small molecules, proteins, polypeptides or peptides and the like.

The term “nucleic acids” or “oligonucleotide” also denotes DNA, cDNA,RNA, siRNA, RNAi, dsRNA and the like, which encode translated anduntranslated regions or inhibits translated or untranslated regions ofstructural genes encoding a peptide or protein or regulatory region. Forexample, a nucleic acid of the invention can include 5′ and 3′untranslated regulatory nucleotide sequences as well as translatedsequences associated with a structural gene. The term “nucleic acids” or“oligonucleotide” or grammatical equivalents as used herein, refers toat least two nucleotides covalently linked together.

Additionally, the term “oligonucleotide” refers to structures includingmodified portions such as modified sugar moieties, modified basemoieties or modified sugar linking moieties. These modified portionsfunction in a manner similar to natural bases, natural sugars andnatural phosphodiester linkages. Accordingly, oligonucleotides may havealtered base moieties, altered sugar moieties or altered inter-sugarlinkages. Modified linkages may be, for example, phosphoramide,phosphorothioate, phosphorodithioate, methyl phosphonate,phosphotriester, phosphoramidate, O-methylphophoroamidite linkages, orpeptide nucleic acid backbones and linkages. Other analogs may includeoligonucleotides with positive backbones, non-ionic backbones andnon-ribose backbones. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of natural ormodified bases, including uracil, adenine, thymine, cytosine, guanine,inosine, xathanine, hypoxathanine, isocytosine, isoguanine, halogentatedbases and the like. Other modifications may include, for example, deazaor aza purines and pyrimidines used in place of natural purine andpyrimidine bases; pyrimidine bases having substituent groups at the 5-or 6-positions, purine bases having altered or replacement substituentgroups at the 2-, 6- or 8-positions, or sugars having substituent groupsat their 2′-position, substitutions for one or more of the hydrogenatoms of the sugar, or carbocyclic or acyclic sugars.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation.

Antisense molecules include oligonucleotides comprising a singe-strandednucleic acid sequence (either RNA or DNA) capable of binding to targetreceptor or ligand mRNA (sense) or DNA (antisense) sequences. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein. Antisense or senseoligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones and wherein such sugar linkages areresistant to endogenous nucleases. Such oligonucleotides with resistantsugar linkages are stable in vivo (i.e., capable of resisting enzymaticdegradation) but retain sequence specificity to be able to bind totarget nucleotide sequences.

RNAi is a phenomenon in which the introduction of dsRNA into a diverserange of organisms and cell types causes degradation of thecomplementary mRNA. In the cell, long dsRNAs are cleaved into short(e.g., 21-25 nucleotide) small interfering RNAs (siRNAs), by aribonuclease. The siRNAs subsequently assemble with protein componentsinto an RNA-induced silencing complex (RISC), unwinding in the process.The activated RISC then binds to complementary transcripts by basepairing interactions between the siRNA antisense strand and the mRNA.The bound mRNA is then cleaved and sequence specific degradation of mRNAresults in gene silencing. As used herein, “silencing” refers to amechanism by which cells shut down large sections of chromosomal DNAresulting in suppressing the expression of a particular gene. The RNAimachinery appears to have evolved to protect the genome from endogenoustransposable elements and from viral infections. Thus, RNAi can beinduced by introducing nucleic acid molecules complementary to thetarget mRNA to be degraded.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties andother moieties that increase affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents, such as ellipticine, and alkylating agents ormetal complexes may be attached to sense or antisense oligonucleotidesto modify binding specificities of the antisense or senseoligonucleotide for the target nucleotide sequence.

A peptide of the invention may be any medically or diagnostically usefulpeptide or protein of small to medium size (i.e. up to about 15 kD, 30kD, 40 kD, 50 kD, 60 kD, 70 kD, 80 kD, 90 kD, 100 kD, for example). Themechanisms of improved polypeptide absorption are described in U.S. Pat.No. 5,661,130 which is hereby incorporated by reference in its entirety.Invention compositions can be mixed with all such peptides, although thedegree to which the peptide benefits are improved may vary according tothe molecular weight and the physical and chemical properties of thepeptide, and the particular surfactant used. Examples of polypeptidesinclude vasopressin, vasopressin polypeptide analogs, desmopressin,glucagon, corticotropin (ACTH), gonadotropin, calcitonin, C-peptide ofinsulin, parathyroid hormone (PTH), growth hormone (HG), human growthhormone (hGH), growth hormone releasing hormone (GHRH), oxytocin,corticotropin releasing hormone (CRH), somatostatin or somatostatinpolypeptide analogs, gonadotropin agonist or gonadotrophin agonistpolypeptide analogs, human atrial natriuretic peptide (ANP), humanthyroxine releasing hormone (TRH), follicle stimulating hormone (FSH),prolactin, insulin, insulin like growth factor-I (IGF-I) somatomedin-C(SM-C), calcitonin, leptin and the leptin derived short peptide OB-3,melatonin, GLP-1 or Glucagon-like peptide-1, GiP, neuropeptide pituitaryadenylate cyclase, GM-1 ganglioside, nerve growth factor (NGF),nafarelin, D-tryp6)-LHRH, FGF, VEGF antagonists, leuprolide, interferon(e.g., α, β, γ) low molecular weight heparin, PYY, LHRH antagonists,Keratinocyte Growth Factor (KGF), Glial-Derived Neurotrophic Factor(GDNF), ghrelin, and ghrelin antagonists. Further, in some aspects, thepeptide or protein is selected from a growth factor, interleukin,polypeptide vaccine, enzyme, endorphin, glycoprotein, lipoprotein, or apolypeptide involved in the blood coagulation cascade.

Other drugs or therapeutic compounds, molecules and/or agents includecompounds or molecules of the central nervous system affectingneurotransmitters or neural ion channels (i.e. antidepressants(bupropion)), selective serotonin 2c receptor agonists, anti-seizureagents (topiramate, zonisamide), some dopamine antagonists, andcannabinoid-1 receptor antagonists (rimonabant)); leptin/insulin/centralnervous system pathway agents (i.e. leptin analogues, leptin transportand/or leptin receptor promoters, ciliary neurotrophic factor (Axokine),neuropeptide Y and agouti-related peptide antagonists,proopiomelanocortin, cocaine and amphetamine regulated transcriptpromoters, alpha-melanocyte-stimulating hormone analogues,melanocortin-4 receptor agonists, protein-tyrosine phosphatase-1Binhibitors, peroxisome proliferator activated receptor-gamma receptorantagonists, short-acting bromocriptine (ergoset), somatostatin agonists(octreotide), and adiponectin); gastrointestinal-neural pathway agents(i.e. agents that increase glucagon-like peptide-1 activity (extendin-4,liraglutide, dipeptidyl peptidase IV inhibitors), protein YY3-36,ghrelin, ghrelin antagonists, amylin analogues (pramlintide)); andcompounds or molecules that may increase resting metabolic rate“selective” beta-3 stimulators/agonist, melanin concentrating hormoneantagonists, phytostanol analogues, functional oils, P57, amylaseinhibitors, growth hormone fragments, synthetic analogues ofdehydroepiandrosterone sulfate, antagonists of adipocyte11B-hydroxysteroid dehydrogenase type 1 activity,corticotropin-releasing hormone agonists, inhibitors of fatty acidsynthesis, carboxypeptidase inhibitors, and gastrointestinal lipaseinhibitors (ATL962).

The therapeutic composition of the invention includes a drug and a drugabsorption enhancing agent, for example, a surfactant. The term“surfactant” is any surface active agent that modifies interfacialtension of water. Typically, surfactants have one lipophilic and onehydrophilic group in the molecule. Broadly, the group includes soaps,detergents, emulsifiers, dispersing and wetting agents, and severalgroups of antiseptics. More specifically, surfactants includestearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride andglycerin monostearate; and hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethylcellulose sodium,methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose andhydroxypropylcellulose.

Preferably, the surfactant of the invention consists of at least onesuitable alkyl glycoside. As used herein, “alkyl glycoside” refers toany sugar joined by a linkage to any hydrophobic alkyl, as is known inthe art. Any “suitable” alkyl glycoside means one that fulfills thelimiting characteristics of the invention, i.e., that the alkylglycoside be nontoxic and nonionic, and that it increases the absorptionof a compound when it is administered with the compound via the ocular,nasal, nasolacrimal, inhalation or pulmonary, oral cavity (sublingual orBuccal cell), or CSF delivery route. Suitable compounds can bedetermined using the methods set forth herein.

Alkyl glycosides of the invention can be synthesized by knownprocedures, i.e., chemically, as described, e.g., in Rosevear et al.,Biochemistry 19:4108-4115 (1980) or Koeltzow and Urfer, J. Am. Oil Chem.Soc., 61:1651-1655 (1984), U.S. Pat. No. 3,219,656 and U.S. Pat. No.3,839,318 or enzymatically, as described, e.g., in Li et al., J. Biol.Chem., 266:10723-10726 (1991) or Gopalan et al., J. Biol. Chem.267:9629-9638 (1992).

Alkyl glycosides of the present invention can include, but are notlimited to: alkyl glycosides, such as octyl-, nonyl-, decyl-, undecyl-,dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-,and octadecyl-α- or β-D-maltoside, -glucoside or -sucroside (synthesizedaccording to Koeltzow and Urfer; Anatrace Inc., Maumee, Ohio;Calbiochem, San Diego, Calif.; Fluka Chemie, Switzerland); alkylthiomaltosides, such as heptyl, octyl, dodecyl-, tridecyI-, andtetradecyl-β-D-thiomaltoside (synthesized according to Defaye, J. andPederson, C., “Hydrogen Fluoride, Solvent and Reagent for CarbohydrateConversion Technology” in Carbohydrates as Organic Raw Materials,247-265 (F. W. Lichtenthaler, ed.) VCH Publishers, New York (1991);Ferenci, T., J. Bacteriol, 144:7-11 (1980)); alkyl thioglucosides, suchas heptyl- or octyl 1-thio α- or β-D-glucopyranoside (Anatrace, Inc.,Maumee, Ohio; see Saito, S. and Tsuchiya, T. Chem. Pharm. Bull.33:503-508 (1985)); alkyl thiosucroses (synthesized according to, forexample, Binder, T. P. and Robyt, J. F., Carbohydr. Res. 140:9-20(1985)); alkyl maltotriosides (synthesized according to Koeltzow andUrfer); long chain aliphatic carbonic acid amides of sucroseβ-amino-alkyl ethers; (synthesized according to Austrian Patent 382,381(1987); Chem. Abstr., 108:114719 (1988) and Gruber and Greber pp.95-116); derivatives of palatinose and isomaltamine linked by amidelinkage to an alkyl chain (synthesized according to Kunz, M.,“Sucrose-based Hydrophilic Building Blocks as Intermediates for theSynthesis of Surfactants and Polymers” in Carbohydrates as Organic RawMaterials, 127-153); derivatives of isomaltamine linked by urea to analkyl chain (synthesized according to Kunz); long chain aliphaticcarbonic acid ureides of sucrose β-amino-alkyl ethers (synthesizedaccording to Gruber and Greber, pp. 95-116); and long chain aliphaticcarbonic acid amides of sucrose β-amino-alkyl ethers (synthesizedaccording to Austrian Patent 382, 381 (1987), Chem. Abstr., 108:114719(1988) and Gruber and Greber, pp. 95-116).

Surfactants of the invention consisting of an alkyl glycoside and/or asucrose ester have characteristic hydrophile-lipophile balance (HLB)numbers, which can be calculated or determined empirically (Schick, M.J. Nonionic Surfactants, p. 607 (New York: Marcel Dekker, Inc. (1967)).The HLB number is a direct reflection of the hydrophilic character ofthe surfactant, i.e., the larger the HLB number, the more hydrophilicthe compound. HLB numbers can be calculated by the formula: (20 times MWhydrophilic component)/(MW hydrophobic component+MW hydrophiliccomponent), where MW=molecular weight (Rosen, M. J., Surfactants andInterfacial Phenomena, pp. 242-245, John Wiley, New York (1978)). TheHLB number is a direct expression of the hydrophilic character of thesurfactant, i.e., the larger the HLB number, the more hydrophilic thecompound. A preferred surfactant has an HLB number of from about 10 to20 and an even more preferred range of from about 11 to 15.

As described above, the hydrophobic alkyl can thus be chosen of anydesired size, depending on the hydrophobicity desired and thehydrophilicity of the saccharide moiety. For example, one preferredrange of alkyl chains is from about 9 to about 24 carbon atoms. An evenmore preferred range is from about 9 to about 16 or about 14 carbonatoms. Similarly, some preferred glycosides include maltose, sucrose,and glucose linked by glycosidic linkage to an alkyl chain of 9, 10, 12,13, 14, 16, 18, 20, 22, or 24 carbon atoms, e.g., nonyl-, decyl-,dodecyl- and tetradecyl sucroside, glucoside, and maltoside, etc. Thesecompositions are nontoxic, since they are degraded to an alcohol and anoligosaccharide, and amphipathic.

The surfactants of the invention can also include a saccharide. As useherein, a “saccharide” is inclusive of monosaccharides, oligosaccharidesor polysaccharides in straight chain or ring forms, or a combinationthereof to form a saccharide chain. Oligosaccharides are saccharideshaving two or more monosaccharide residues. The saccharide can bechosen, for example, from any currently commercially availablesaccharide species or can be synthesized. Some examples of the manypossible saccharides to use include glucose, maltose, maltotriose,maltotetraose, sucrose and trehalose. Preferable saccharides includemaltose, sucrose and glucose.

The surfactants of the invention can likewise consist of a sucroseester. As used herein, “sucrose esters” are sucrose esters of fattyacids and is a complex of sucrose and fatty acid. Sucrose esters cantake many forms because of the eight hydroxyl groups in sucroseavailable for reaction and the many fatty acid groups, from acetate onup to larger, more bulky fatty acids that can be reacted with sucrose.This flexibility means that many products and functionalities can betailored, based on the fatty acid moiety used. Sucrose esters have foodand non-food uses, especially as surfactants and emulsifiers, withgrowing applications in pharmaceuticals, cosmetics, detergents and foodadditives. They are biodegradable, non-toxic and mild to the skin.

The surfactants of the invention have a hydrophobic alkyl group linkedto a hydrophilic saccharide. The linkage between the hydrophobic alkylgroup and the hydrophilic saccharide can include, among otherpossibilities, a glycosidic, thioglycosidic (Horton), amide(Carbohydrates as Organic Raw Materials, F. W. Lichtenthaler ed., VCHPublishers, New York, 1991), ureide (Austrian Pat. 386,414 (1988); Chem.Abstr. 110:137536p (1989); see Gruber, H. and Greber, G., “ReactiveSucrose Derivatives” in Carbohydrates as Organic Raw Materials, pp.95-116) or ester linkage (Sugar Esters: Preparation and Application, J.C. Colbert ed., (Noyes Data Corp., New Jersey), (1974)). Further,preferred glycosides can include maltose, sucrose, and glucose linked byglycosidic linkage to an alkyl chain of about 9-16 carbon atoms, e.g.,nonyl-, decyl-, dodecyl- and tetradecyl sucroside, glucoside, andmaltoside. Again, these compositions are amphipathic and nontoxic,because they degrade to an alcohol and an oligosaccharide.

The above examples are illustrative of the types of glycosides to beused in the methods claimed herein, but the list is not exhaustive.Derivatives of the above compounds which fit the criteria of the claimsshould also be considered when choosing a glycoside. All of thecompounds can be screened for efficacy following the methods taughtherein and in the examples.

The compositions of the present invention can be administered in aformat selected from the group consisting of a tablet, a capsule, asuppository, a drop, a spray, an aerosol and a sustained release ordelayed burst format. The spray and the aerosol can be achieved throughuse of an appropriate dispenser. The sustained release format can be anocular insert, erodible microparticulates, swelling mucoadhesiveparticulates, pH sensitive microparticulates, nanoparticles/latexsystems, ion-exchange resins and other polymeric gels and implants(Ocusert, Alza Corp., California; Joshi, A., S. Ping and K. J.Himmelstein, Patent Application WO 91/19481). These systems maintainprolonged drug contact with the absorptive surface preventing washoutand nonproductive drug loss. The prolonged drug contact is non-toxic tothe skin and mucosal surfaces.

The surfactant compositions of the invention are stable. For example,Baudys et al. in U.S. Pat. No. 5,726,154 show that calcitonin in anaqueous liquid composition comprising SDS (sodium dodecyl sulfate, asurfactant) and an organic acid is stable for at least 6 months.Similarly, the surfactant compositions of the present invention haveimproved stabilizing characteristics when admixed with a drug. Noorganic acid is required in these formulations. For example, thecomposition of the invention maintains the stability of proteins andpeptide therapeutics for about 6 months, or more, when maintained atabout 4° C. to 25° C.

The stability of the surfactant compositions are, in part, due to theirhigh no observable adverse effect level (NOAEL). The EnvironmentalProtection Agency (EPA) defines the no observable adverse effect level(NOAEL) as the exposure level at which there are no statistically orbiologically significant increases in the frequency or severity ofadverse effects between the exposed population and its appropriatecontrol. Hence, the term, “no observable adverse effect level” (orNOAEL) is the greatest concentration or amount of a substance, found byexperiment or observation, which causes no detectable adverse alterationof morphology, functional capacity, growth, development, or life span ofthe target organism under defined conditions.

The Food and Agriculture Organization (FAO) of the United Nations of theWorld Health Organization (WHO) has shown that some alkyl glycosideshave very high NOAELs, allowing for increased consumption of these alkylglycosides without any adverse effect. This report can be found on theworld wide web at inchem.org/documents/jecfa/jecmono/v10je11.htm. Forexample, the NOAEL for sucrose dodecanoate, a sucrose ester used in foodproducts, is about 20-30 grams/kilogram/day, e.g. a 70 kilogram person(about 154 lbs.) can consume about 1400-2100 grams (or about 3 to 4.6pounds) of sucrose dodecanoate per day without any observable adverseeffect. Typically, an acceptable daily intake for humans is about 1% ofthe NOAEL, which translates to about 14-21 grams, or 14 millionmicrograms to 21 million micrograms, per day, indefinitely. Definitionsof NOAELs and other related definitions can be found on the world wideweb at epa.gov/OCEPAterms. Thus, although some effects may be producedwith alkyl glycoside levels anticipated in the present invention, thelevels are not considered adverse, or precursors to adverse effects.

Accordingly, a subject treated with surfactant compositions of theinvention having at least one alkyl glycoside, e.g. tetradecylmaltoside(TDM; or Intravail A), at a concentration of about 0.125% by weight ofalkyl glycoside two times per day, or three times per day, or moredepending on the treatment regimen consumes about 200 to 300 microgramsper day total of TDM. So, the effective dose of the TDM is at least1000× fold lower than (i.e., 1/1000) of the NOAEL, and falls far below1% of the NOAEL, which is the acceptable daily intake; or in this caseabout 1/50,000 of the acceptable daily intake. Stated another way, alkylglycosides of the present invention have a high NOAEL, such that theamount or concentration of alkyl glycosides used in the presentinvention do not cause an adverse effect and can be safely consumedwithout any adverse effect.

The surfactant compositions of the invention are also stable becausethey are physiologically non-toxic and non-irritants. The term,“nontoxic” means that the alkyl glycoside molecule has a sufficientlylow toxicity to be suitable for human administration and consumption.Preferred alkyl glycosides are non-irritating to the tissues to whichthey are applied. Any alkyl glycoside used should be of minimal or notoxicity to the cell, such that it does not cause damage to the cell.Yet, toxicity for any given alkyl glycoside may vary with theconcentration of alkyl glycoside used. It is also beneficial if thealkyl glycoside chosen is metabolized or eliminated by the body and ifthis metabolism or elimination is done in a manner that will not beharmfully toxic. The term, “non-irritant” means that the agent does notcause inflammation following immediate, prolonged or repeated contactwith the skin surface or mucous membranes.

Moreover, one embodiment of the surfactant compositions, in particular,the sucrose esters, serve as anti-bacterial agents. An agent is an“anti-bacterial” agent or substance if the agent or its equivalentdestroy bacteria, or suppress bacterial growth or reproduction. Theanti-bacterial activity of sucrose esters and their fatty acids havebeen reported. Tetsuaki et al. (1997) “Lysis of Bacillus subtilis cellsby glycerol and sucrose esters of fatty acids,” Applied andEnvironmental Microbiology, 53(3):505-508. Watanabe et al. (2000)describe that galactose and fructose laureates are particularlyeffective carbohydrate monoesters. Watanabe et al., (2000)“Antibacterial carbohydrate monoesters suppressing cell growth ofStreptococcus mutan in the presence of sucrose,” Curr Microbiol 41(3):210-213. Hence, the present invention is not limited to the sucroseester described herein, but encompasses other carbohydrate esters,including galactose and fructose esters, that suppress bacterial growthand reproduction.

In general, all useful antimicrobial agents are toxic substances. SeeSutton and Porter (2002), “Development of the antimicrobialeffectiveness test as USP Chapter <51>,” 56(6): 300-311, which isincorporated herein by reference in its entirety. For example, commonlyused antimicrobial agents such as benzalkonium chloride are highly toxicas demonstrated by electron micrograph studies in which significantdisruption of the mucociliary surfaces are observed at concentrations ofbenzalkonium far below what is commonly used in intranasal formulations.See for example Sebahattin Cüreoglu, Murat Akkus, Üstün Osma, MehmetYaldiz, Faruk Oktay, Belgin Can, Cengiz Güven, Muhammet Tekin, and FarukMeriç (2002), “The effect of benzalkonium chloride an electronmicroscopy study,” Eur Arch Otorhinolaryngol 259:362-364.

The surfactant compositions of the invention are typically present at alevel of from about 0.01% to 20% by weight. More preferred levels ofincorporation are from about 0.01% to 5% by weight, from about 0.01% to2% by weight, from about 0.01% to 1%, most preferably from about 0.01%to 0.125% by weight. The surfactant is preferably formulated to becompatible with other components present in the composition. In liquid,or gel, or capsule, or injectable, or spray compositions the surfactantis most preferably formulated such that it promotes, or at least doesnot degrade, the stability of any protein or enzyme in thesecompositions. Further, the invention optimizes the concentration bykeeping the concentration of absorption enhancer as low as possible,while still maintaining the desired effect.

The compositions of the invention when administered to the subject,yield enhanced mucosal delivery of the biologically active compound(s),or drug, with a peak concentration (or Cmax) of the compound(s) in atissue, or fluid, or in a blood plasma of the subject that is about 15%,20%, or 50% or greater as compared to a Cmax of the compound(s) in atissue (e.g. CNS), or fluid, or blood plasma following intramuscularinjection of an equivalent concentration of the compound(s) to thesubject.

The measure of how much of the drug or compound(s) reaches thebloodstream in a set period of time, e.g. 24 hours can also becalculated by plotting drug blood concentration at various times duringa 24-hour or longer period and then measuring the area under the curve(AUC) between 0 and 24 hours. Similarly, a measure of drug efficacy canalso be determined from a time to maximal concentration (tmax) of thebiologically active compound(s) in a tissue (e.g. CNS) or fluid or inthe blood plasma of the subject between about 0.1 to 1.0 hours. Thetherapeutic compositions of the invention increase the speed of onset ofdrug action (i.e., reduce Tmax) by a factor of about 1.5-fold to 2-fold.

Also, the therapeutic compositions or formulations of the invention canbe administered or delivered to a subject in need systemically orlocally. Suitable routes may, for example, include oral, ocular, nasal,nasolacrimal, inhalation or pulmonary, oral cavity (sublingual or Buccalcell), transmucosal administration, vaginal, rectal, parenteraldelivery, including intramuscular, subcutaneous, intravenous,intraperitoneal, or CSF delivery. Moreover, the mode of delivery e.g.liquid, gel, tablet, spray, etc. will also depend on the method ofdelivery to the subject.

Additionally, the therapeutic compositions of the invention can consistof a pharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” is an aqueous or non-aqueous agent, for example alcoholic oroleaginous, or a mixture thereof, and can contain a surfactant,emollient, lubricant, stabilizer, dye, perfume, preservative, acid orbase for adjustment of pH, a solvent, emulsifier, gelling agent,moisturizer, stabilizer, wetting agent, time release agent, humectant,or other component commonly included in a particular form ofpharmaceutical composition. Pharmaceutically acceptable carriers arewell known in the art and include, for example, aqueous solutions suchas water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of the specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Apharmaceutically acceptable carrier can also be selected from substancessuch as distilled water, benzyl alcohol, lactose, starches, talc,magnesium stearate, polyvinylpyrrolidone, alginic acid, colloidalsilica, titanium dioxide, and flavoring agents.

Additionally, to decrease susceptibility of alkyl saccharides orsaccharide alkyl esters to hydrolytic cleavage of the drug, variousoxygen atoms within the drugs can be substituted for by sulfur (Defaye,J. and Gelas, J. in Studies in Natural Product Chemistry(Atta-ur-Rahman, ed.) Vol. 8, pp. 315-357, Elsevier, Amsterdam, 1991).For example, the heteroatom of the sugar ring can be either oxygen orsulfur, or the linkage between monosaccharides in an oligosaccharide canbe oxygen or sulfur (Horton, D. and Wander, J. D., “Thio Sugars andDerivatives,” The Carbohydrates: Chemistry and Biochemistry, 2d. Ed.Vol. IB, (W. Reyman and D. Horton eds.), pp. 799-842, (Academic Press,New York), (1972)). Oligosaccharides can have either α (alpha) or β(beta) anomeric configuration (see Pacsu, E., et al. in Methods inCarbohydrate Chemistry (R. L. Whistler, et al., eds.) Vol. 2, pp.376-385, Academic Press, New York 1963).

A composition of the invention can be prepared in tablet form by mixinga therapeutic agent or drug and one alkyl glycoside and/or saccharidealkyl ester according to the invention, and an appropriatepharmaceutical carrier or excipient, for example mannitol, corn starch,polyvinylpyrrolidone or the like, granulating the mixture and finallycompressing it in the presence of a pharmaceutical carrier such as cornstarch, magnesium stearate or the like. If necessary, the formulationthus prepared may include a sugar-coating or enteric coating or coveredin such a way that the active principle is released gradually, forexample, in the appropriate pH medium.

The term “enteric coating,” is a polymer encasing, surrounding, orforming a layer, or membrane around the therapeutic composition or core.Also, the enteric coating can contain a drug which is compatible orincompatible with the coating. One tablet composition may include anenteric coating polymer with a compatible drug which dissolves orreleases the drug at higher pH levels (e.g., pH greater than 4.0,greater than 4.5, greater than 5.0 or higher) and not at low pH levels(e.g., pH 4 or less); or the reverse.

In a preferred embodiment, the dose dependent release form of theinvention is a tablet comprising:

(a) a core comprising:

(i) a therapeutic agent or drug;

(ii) a surfactant comprising at least one alkyl glycoside and/orsaccharide alkyl ester; and

(b) at least one membrane coating surrounding the core, wherein thecoating is an impermeable, permeable, semi-permeable or porous coatingand becomes more permeable or porous upon contacting an aqueousenvironment of a defined pH.

The term “membrane” is synonymous with “coating,” or equivalentsthereof. The terms are used to identify a region of a medicament, forexample, a tablet, that is impermeable, permeable, semi-permeable orporous to an aqueous solution(s) or bodily fluid(s), and/or to thetherapeutic agent(s) or drug(s) encapsulated therein. If the membrane ispermeable, semi-permeable or porous to the drug, the drug can bereleased through the openings or pores of the membrane in solution or invivo. The porous membrane can be manufactured mechanically (e.g.,drilling microscopic holes or pores in the membrane layer using alaser), or it can be imparted due to the physiochemical properties ofthe coating polymer(s). Membrane or coating polymers of the inventionare well known in the art, and include cellulose esters, cellulosediesters, cellulose triesters, cellulose ethers, cellulose ester-ether,cellulose acylate, cellulose diacylate, cellulose triacylate, celluloseacetate, cellulose diacetate, cellulose triacetate, cellulose acetatepropionate, and cellulose acetate butyrate. Other suitable polymers aredescribed in U.S. Pat. Nos. 3,845,770, 3,916,899, 4,008,719, 4,036,228and 4,11210 which are incorporated herein by reference.

Further, the enteric coating according to the invention can include aplasticizer, and a sufficient amount of sodium hydroxide (NaOH) toeffect or adjust the pH of the suspension in solution or in vivo.Examples of plasticizers include triethyl citrate, triacetin, tributylsebecate, or polyethylene glycol. Other alkalizing agents, includingpotassium hydroxide, calcium carbonate, sodium carboxymethylcellulose,magnesium oxide, and magnesium hydroxide can also be used to effect oradjust the pH of the suspension in solution or in vivo.

Accordingly, in one embodiment, an enteric coating can be designed torelease a certain percentage of a drug or drugs in certain mediums witha certain pH or pH range. For example, the therapeutic composition ofthe invention may include at least one enteric coating encasing orprotecting at least one drug which is chemically unstable in an acidicenvironment (e.g., the stomach). The enteric coating protects the drugfrom the acidic environment (e.g., pH<3), while releasing the drug inlocations which are less acidic, for example, regions of the small andlarge intestine where the pH is 3, or 4, or 5, or greater. A medicamentof this nature will travel from one region of the gastrointestinal tractto the other, for example, it takes about 2 to about 4 hours for a drugto move from the stomach to the small intestine (duodenum, jejunum andileum). During this passage or transit, the pH changes from about 3(e.g., stomach) to 4, or 5, or to about a pH of 6 or 7 or greater. Thus,the enteric coating allows the core containing the drug to remainsubstantially intact, and prevents premature drug release or the acidfrom penetrating and de-stabilizing the drug.

Examples of suitable enteric polymers include but are not limited tocellulose acetate phthalate, hydroxypropylmethylcellulose phthalate,polyvinylacetate phthalate, methacrylic acid copolymer, shellac,cellulose acetate trimellitate, hydroxypropylmethylcellulose acetatesuccinate, hydroxypropylmethylcellulose phthalate, cellulose acetatephthalate, cellulose acetate succinate, cellulose acetate malate,cellulose benzoate phthalate, cellulose propionate phthalate,methylcellulose phthalate, carboxymethylethylcellulose,ethylhydroxyethylcellulose phthalate, shellac, styrene-acrylic acidcopolymer, methyl acrylate-acrylic acid copolymer, methylacrylate-methacrylic acid copolymer, butyl acrylate-styrene-acrylic acidcopolymer, methacrylic acid-methyl methacrylate copolymer, methacrylicacid-ethyl acrylate copolymer, methyl acrylate-methacrylic acid-octylacrylate copolymer, vinyl acetate-maleic acid anhydride copolymer,styrene-maleic acid anhydride copolymer, styrene-maleic acid monoestercopolymer, vinyl methyl ether-maleic acid anhydride copolymer,ethylene-maleic acid anhydride copolymer, vinyl butyl ether-maleic acidanhydride copolymer, acrylonitrile-methyl acrylate-maleic acid anhydridecopolymer, butyl acrylate-styrene-maleic acid anhydride copolymer,polyvinyl alcohol phthalate, polyvinyl acetal phthalate, polyvinylbutylate phthalate and polyvinyl acetoacetal phthalate, or combinationsthereof. One skilled in the art will appreciate that other hydrophilic,hydrophobic and enteric coating polymers may be readily employed, singlyor in any combination, as all or part of a coating according to theinvention.

The therapeutic compositions of the invention in the form of a tabletcan have a plurality of coatings, for example, a hydrophilic coating(e.g., hydroxypropylmethyl-cellulose), and/or a hydrophobic coating(e.g., alkylcelluloses), and/or an enteric coating. For example, thetablet core can be encases by a plurality of the same type of coating,or a plurality of different types of coating selected from ahydrophilic, hydrophobic or enteric coating. Hence, it is anticipatedthat a tablet can be designed having at least one, but can have morethan one layer consisting of the same or different coatings dependent onthe target tissue or purpose of the drug or drugs. For example thetablet core layer may have a first composition enclosed by a firstcoating layer (e.g. hydrophilic, hydrophobic, or enteri-coating), and asecond same or different composition or drug having the same ordifferent dosage can be enclosed in second coating layer, etc. Thislayering of various coatings provides for a first, second, third, ormore gradual or dose dependent release of the same or different drugcontaining composition.

In a preferred embodiment, a first dosage of a first composition of theinvention is contained in a tablet core and with an enteric-coating suchthat the enteric-coating protects and prevents the composition containedtherein from breaking down or being released into the stomach. Inanother example, the first loading dose of the therapeutic compositionis included in the first layer and consists of from about 10% to about40% of the total amount of the total composition included in theformulation or tablet. In a second loading dose, another percentage ofthe total dose of the composition is released. The inventioncontemplates as many time release doses as is necessary in a treatmentregimen. Thus, in certain aspects, a single coating or plurality ofcoating layers is in an amount ranging from about 2% to 6% by weight,preferably about 2% to about 5%, even more preferably from about 2% toabout 3% by weight of the coated unit dosage form.

Accordingly, the composition preparations of the invention make itpossible for contents of a hard capsule or tablet to be selectivelyreleased at a desired site the more distal parts of thegastro-intestinal tract (e.g. small and large intestine) by selectingthe a suitable pH-soluble polymer for a specific region. Mechanicalexpulsion of the composition preparations may also be achieved byinclusion of a water absorbing polymer that expands upon waterabsorption within a hard semi-permeable capsule thus expellingcomposition through an opening in the hard capsule.

Drugs particularly suited for dose dependent time release include butare not limited to insulin like growth factor-I (IGF-I), somatomedin-C(SM-C; diabetes, nerve function, renal function), insulin (diabetes),calcitonin (osteoporosis), leptin (obesity; infertility), leptin derivedshort peptide (OB-3), hGH (AIDs wasting, dwarfism), human parathyroidhormone (PTH) (osteoporosis), melatonin (sleep), GLP-1 or Glucagon-likepeptide-1 (diabetes), GiP (diabetes), pituitary adenylatecyclase-activating polypeptide (PACAP) and islet function (diabetes),GM-1 ganglioside, (Alzheimers), nerve growth factor (NGF), (Alzheimers),nafarelin (endometriosis), Synarel® (nafarelin acetate nasal solution),(D-tryp6)-LHRH (fertility), FGF (duodenal ulcer, macular degeneration,burns, wounds, spinal cord injuries, repair of bone and cartilagedamage), VEGF antagonists (to block the receptor), VEGF (agonist)neonatal distress syndrome; ALS), leuprolide (prostate and breastcancer), interferon-alpha (chronic hepatitis C), low molecular weightheparin (blood clotting, deep vein thrombosis), PYY (obesity), LHRHantagonists (fertility), LH (luteinizing hormone), ghrelin antagonists(obesity), KGF (Parkinson's), GDNF (Parkinsons), G-CSF (erythropoiesisin cancer), Imitrex (migraine), Integrelin (anticoagulation), Natrecor®(congestive heart failure), human B-type natriuretic peptide (hBNP),SYNAREL® (Searl; nafarelin acetate nasal solution), Sandostatin (growthhormone replacement), Forteo (osteoporosis), DDAVP® Nasal Spray(desmopressin acetate), Cetrotide® (cetrorelix acetate for injection),Antagon™ (ganirelix acetate), Angiomax (bivalirudin; thrombininhibitor), Accolate® (zafirlukast; injectable), Exendin-4 (Exanatide;diabetes), SYMLIN® (pramlintide acetate; synthetic amylin; diabetes),desmopressin, glucagon, ACTH (corticotrophin), C-peptide of insulin,GHRH and analogs (GnRHa), growth hormone releasing hormone, oxytocin,corticotropin releasing hormone (CRH), atrial natriuretic peptide (ANP),thyroxine releasing hormone (TRHrh), follicle stimulating hormone (FSH),prolactin, tobramycin ocular (corneal infections), Vasopressin,desmopresin, Fuzeon (Roche; HIV fusion inhibitor MW 4492), andEptifibatide.

Further, it will be understood by one skilled in the art, that thespecific dose level and frequency of dosage for any particular subjectin need of treatment may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the host undergoing therapy.

It has been shown that alkyl glycosides, particularly alkylmaltosidesand more specifically, dodecylmaltoside (DDM) and tetradecylmaltoside(TDM), stabilize insulin in solution and prevent aggregation of thepeptide. Hovgaard et al., “Insulin Stabilization and GI absorption,” J.Control. Rel., 19 (1992) 458-463, cited in Hovgaard et al.,“Stabilization of insulin by alkylmaltosides: A spectroscopicevaluation,” Int. J. Pharmaceutics 132 (1996) 107-113 (hereinafter,“Hovgaard-1”). Further, Hovgaard-1 shows that even after 57 days, theDDM-insulin complex remained stable and possessed nearly full biologicalactivity. It is postulated that the stability of the complex is due tothe length of the alkyl group (number of carbon atoms) and the higherratio of DDM to insulin ratio the better (e.g. 4:1 and 16:1; see FIG. 1in Hovgaard 1). However, according to Hovgaard-1, although theDDM-insulin complex was stable, the same stability was not shown forother maltosides. Yet, in a related study, Hovgaard et al. (1996)demonstrated that when DDM-insulin was orally administered to animals invivo, bioavailability of the complex was weak (e.g. 0.5%-1%bioavailability). Hovgaard et al., “Stabilization of insulin byalkylmaltoside. B. Oral absorption in vivo in rats,” Int. J.Pharmaceutics 132 (1996) 115-121 (Hovgaard-2). Hence, an improved aspectof the invention is that the surfactant increases the bioavailability ofa drug to the target tissues, organs, system etc., as well as increasedrug stability.

Accordingly, one aspect of the invention is to provide therapeuticcompositions having at least one drug and one surfactant, wherein thesurfactant further consists of at least one alkyl glycoside and/orsaccharide alkyl ester formulation which enhances the bioavailability ofthe drug. Determining the bioavailability of drug formulations isdescribed herein. As used herein, “bioavailability” is the rate andextent to which the active substance, or moiety, which reaches thesystemic circulation as an intact drug. The bioavailability of any drugwill depend on how well is adsorbed and how much of it escapes beingremoved from the liver.

To determine absolute bioavailability, the tested drug and mode ofadministration is measured against an intravenous reference dose. Thebioavailability of the intravenous dose is 100% by definition. Forexample, animals or volunteering humans are given an intravenousinjections and corresponding oral doses of a drug. Urinary or plasmasamples are taken over a period of time and levels of the drug over thatperiod of time are determined.

The areas under the curve (AUC), of the plasma drug concentration versustime curves, are plotted for both the intravenous and the oral doses,and calculation of the bioavailability of both formulations is by simpleproportion. For example, if the same intravenous and oral doses aregiven, and the oral AUC is 50% of the intravenous AUC, thebioavailability of the oral formulation is 50%. Note that thebioavailability of any drug is due to many factors including incompleteabsorption, first pass clearance or a combination of these (discussedmore below). Further, the peak concentration (or C_(max)) of the plasmadrug concentration is also measured to the peak concentration (C_(max))of the plasma drug concentration following intramuscular (IM) injectionof an equivalent concentration the drug. Moreover, the time to maximalconcentration (or t_(max)) of the plasma drug is about 0.1 to 1.0 hours.

To determine the relative bioavailability of more than one formulationof a drug (e.g. an alkyl glycoside or saccharide alkyl ester drugformulation), bioavailability of the formulations are assessed againsteach other as one or both drugs could be subject to first pass clearance(discussed more below) and thus undetected. For example, a first oralformulation is assessed against a second oral formulation. The secondformulation is used as a reference to assess the bioavailability of thefirst. This type of study provides a measure of the relative performanceof two formulations in getting a drug absorbed.

Bioavailabilities of drugs are inconsistent and vary greatly from onedrug to the next. For example, the bioavailability of MIACALCIN® (salmoncalcitonin from Novartis) nasal spray, a prescription medication for thetreatment of postmenopausal osteoporosis in women, has a meanbioavailability of about 3% (range is 0.3%-30.6%; see FIG. 1). TheMIACALCIN® product information sheet can be found on the world wide webat miacalcin.com/info/howWorks/index.jsp anddrugs.com/PDR/Miacalcin_Nasal_Spray.html. The data on MIACALCIN®, whichwas obtained by various investigators using different methods and humansubjects, show great variability in the drug's bioavailability, e.g. innormal volunteers only ˜3% of the nasally administered dose isbioavailable, as compared to the same dose administered by intramuscularinjection (MIACALCIN® product insert). This represents two orders of amagnitude in variability and is undesirable to the consumer.

Poor bioavailability of a drug can also be observed in NASCOBAL®(Nastech), or cyanocobalamin, which is used for the treatment andmaintenance of the hematologic status of patients who are in remissionfollowing intramuscular vitamin B₁₂ therapies. The gel formulation wasadministered intranasally and the bioavailability of B₁₂ was compared tointramuscular B₁₂ injections. The peak concentrations of B₁₂ (or theTmax) was reached in 1-2 hours after intranasal administration, andrelative to the intramuscular injection, the bioavailability of B₁₂nasal gel was found to be about 8.9% (90% confidence intervals, 7.1% to11.2%).

The alkyl glycosides or sucrose esters of the present invention includeany compounds now known or later discovered. Drugs which areparticularly well suited for admixture with the alkyl glycosides and/orsaccharide alkyl esters of the invention are those that are difficult toadminister by other methods, e.g. drugs that are degraded in thegastrointestinal (GI) tract or those that are not absorbed well from theGI tract, or drugs that can be self-administered via the ocular, nasal,nasolacrimal, inhalation, or CSF delivery route instead of traditionalmethods such as injection. Some specific examples include peptides,polypeptides, proteins, nucleic acids and other macromolecules, forexample, peptide hormones, such as insulin and calcitonin, enkephalins,glucagon and hypoglycemic agents such as tolbutamide and glyburide, andagents which are poorly absorbed by enteral routes, such asgriseofulvin, an antifungal agent. Other compounds include, for example,nicotine, interferon (e.g., alpha, beta, gamma), PYY, GLP-1, syntheticexendin-4 (Exenatide), parathyroid hormone, and human growth hormone orother low molecular weight peptides and proteins.

Alternatively, bioavailability of a drug can be determined by measuringthe levels of the drug's first pass clearance by the liver. Alkylglycosides and/or saccharide alkyl ester compositions of the inventionadministered intranasally or via oral cavity (sublingual or Buccal cell)do not enter the hepatic portal blood system, thereby avoiding firstpass clearance by the liver. Avoiding first past clearance of theseformulations by the liver is described herein. The term, “first passliver clearance” is the extent to which the drug is removed by the liverduring its first passage in the portal blood through the liver to thesystemic circulation. This is also called first pass metabolism or firstpass extraction.

The two major routes of drug elimination from the body are excretion bythe kidneys whereby the drug is unchanged; and elimination by the liver,whereby the drug is metabolized. The balance between these two routesdepends on the relative efficiency of the two processes. The presentinvention describes herein elimination by the liver or liver clearance.First pass liver clearance is described by Birkett et al (1990 and1991), which is incorporated by reference in its entirety. Birkett etal., Aust Prescr, 13 (1990):88-9; and Birkett et al., Austra Prescr14:14-16 (1991).

Blood carrying drug from the systemic circulation enter the liver viathe portal vein, and the liver in turn extracts a certain percentage orratio (i.e. 0.5 or 50%) of that drug. The remainder left over (i.e. 0.2or 20%) re-enters the systemic circulation via the hepatic vein. Thisrate of clearance of the drug is called the hepatic extraction ratio. Itis the fraction of the drug in the blood which is irreversibly removed(or extracted) during the first pass of the blood through the liver. Ifno drug is extracted, the hepatic extraction ratio is zero. Conversely,if the drug is highly extracted in the first pass through the liver, thehepatic extraction ratio may be as high as 100% or 1.0. In general,clearance of the drug by the liver depends then on the rate of deliveryof that drug to the liver (or the hepatic blood flow), and on theefficiency of removal of that drug (or the extraction ratio).

Therefore, the net equation used to determine hepatic clearance is:(hepatic clearance−blood flow)=(unbound fraction*intrinsicclearance)/blood flow+(unbound fraction*intrinsic clearance)  (1)

The “unbound fraction” of drug is dependent on how tightly the drug isbound to proteins and cells in the blood. In general, it is only thisunbound (or free) drug which is available for diffusion from the bloodinto the liver cell. In the absence of hepatic blood flow and proteinbinding, the “intrinsic clearance” is the ability of the liver to remove(or metabolize) that drug. In biochemical terms, it is a measure ofliver enzyme activity for a particular drug substrate. Again, althoughintrinsic clearance can be high, drugs cannot be cleared more rapidlythan that presented to the liver. In simple terms, there are twosituations: where liver enzyme activity is very high or very low (i.e.high extraction ratio or low extraction ratio).

When liver enzyme activity is low, the equation simplifies to:hepatic clearance=unbound fraction*intrinsic clearance  (2)

Clearance then is independent of blood flow, but instead dependsdirectly on the degree of protein binding in the blood and the activityof drug metabolizing enzymes towards that drug.

In contrast, when liver enzyme activity is high, the equation is:hepatic clearance=liver blood flow  (3)

In this scenario, because the enzymes are so active the liver removesmost of the drug presented to it and the extraction ratio is high. Thus,the only factor determining the actual hepatic clearance is the rate ofsupply of drug to the liver (or hepatic blood flow).

First pass liver clearance is important because even small changes inthe extraction of drugs can cause large changes in bioavailability. Forexample, if the bioavailability of drug A by oral administration is 20%by the time it reaches the systemic circulation, and the same drug A byintravenous administration is 100%, absent no other complicatingfactors, the oral dose will therefore have to be 5 times the intravenousdose to achieve similar plasma concentrations.

Secondly, in some instances where liver enzyme activity is very high,drug formulations should be designed to have the drug pass directlythrough to the systemic circulation and avoid first pass liver clearanceall together. For example, drugs administered intranasally, sublingual,buccal, rectal, vagina, etc. directly enter the systemic circulation anddo not enter the hepatic portal blood circulation to be partially orfully extracted by the liver. Alternatively, where drugs cannot beadministered by the above means, a tablet with at least oneenteric-coating layer to prevent release of the drug in the stomach(i.e. highly acidic environment) is provided. Thus, an objective of theinvention is to administer drugs using these alternative routes.

Additionally, first pass liver clearance is an important factor becausemany patients are on more than one drug regimen, and this may cause druginteractions which increase or decrease liver enzyme activity; therebyincreasing or decreasing metabolism (increasing or decreasing thehepatic extraction ratio) of the drug of interest.

Hence, therapeutic compositions of the invention can be administereddirectly to the systemic circulatory system and avoid first pass liverclearance. Avoiding first pass clearance assures that more of the drugwill be available to the system. Stated another way, by avoiding firstpass liver clearance, the bioavailability of the drug is increased.

The present invention also relates to methods of increasing absorptionof a low molecular compound into the circulatory system of a subjectcomprising administering via the oral, ocular, nasal, nasolacrimal,inhalation, or the CSF delivery route the compound and an absorptionincreasing amount of a suitable nontoxic, nonionic alkyl glycosidehaving a hydrophobic alkyl joined by a linkage to a hydrophilicsaccharide.

The composition formulation is appropriately selected according to theadministration route, such as oral administration (oral preparation),external administration (e.g., ointment), injection (preparations forinjection), and mucosal administration (e.g., buccal and suppository)etc. For example, excipients (e.g., starch, lactose, crystallinecellulose, calcium lactate, magnesium aluminometasilicate and anhydroussilicate), disintegrators (e.g., carboxymethylcellulose and calciumcarboxymethylcellulose), lubricants (e.g., magnesium stearate and talc),coating agents (e.g., hydroxyethylcellulose), and flavoring agents canbe used for oral and mucosal formulations; whereas, solubilizers andauxiliary solubilizers capable of forming aqueous injections (e.g.,distilled water for injection, physiological saline and propyleneglycol), suspending agents (e.g., surfactant such as polysorbate 80), pHregulators (e.g., organic acid and metal salt thereof) and stabilizersare used for injections; and aqueous or oily solubilizers and auxiliarysolubilizers (e.g., alcohols and fatty acid esters), tackifiers (e.g.,carboxy vinyl polymer and polysaccharides) and emulsifiers (e.g.,surfactant) are used for external agents. The drug and the alkylglycoside can be admixed, mixed, or blended along with the aboveexcipients, disintegrators, coating polymers, solubilizers, suspendingagents, etc., prior to administration, or they can be administeredsequentially, in either order. It is preferred that they be mixed priorto administration.

The term, “mucosal delivery-enhancing agent” includes agents whichenhance the release or solubility (e.g., from a formulation deliveryvehicle), diffusion rate, penetration capacity and timing, uptake,residence time, stability, effective half-life, peak or sustainedconcentration levels, clearance and other desired mucosal deliverycharacteristics (e.g., as measured at the site of delivery, or at aselected target site of activity such as the bloodstream or centralnervous system) of a compound(s) (e.g., biologically active compound).Enhancement of mucosal delivery can occur by any of a variety ofmechanisms, including, for example, by increasing the diffusion,transport, persistence or stability of the compound, increasing membranefluidity, modulating the availability or action of calcium and otherions that regulate intracellular or paracellular permeation,solubilizing mucosal membrane components (e.g., lipids), changingnon-protein and protein sulfhydryl levels in mucosal tissues, increasingwater flux across the mucosal surface, modulating epithelial junctionphysiology, reducing the viscosity of mucus overlying the mucosalepithelium, reducing mucociliary clearance rates, and other mechanisms.

Exemplary mucosal delivery enhancing agents include the following agentsand any combinations thereof:

-   -   (a) an aggregation inhibitory agent;    -   (b) a charge-modifying agent;    -   (c) a pH control agent;    -   (d) a degradative enzyme inhibitory agent;    -   (e) a mucolytic or mucus clearing agent;    -   (f) a ciliostatic agent;    -   (g) a membrane penetration-enhancing agent selected from:        -   (i) a surfactant; (ii) a bile salt; (ii) a phospholipid            additive, mixed micelle, liposome, or carrier; (iii) an            alcohol; (iv) an enamine; (v) an NO donor compound; (vi) a            long-chain amphipathic molecule; (vii) a small hydrophobic            penetration enhancer; (viii) sodium or a salicylic acid            derivative; (ix) a glycerol ester of acetoacetic acid; (x) a            cyclodextrin or beta-cyclodextrin derivative; (xi) a            medium-chain fatty acid; (xii) a chelating agent; (xiii) an            amino acid or salt thereof; (xiv) an N-acetylamino acid or            salt thereof; (xv) an enzyme degradative to a selected            membrane component; (ix) an inhibitor of fatty acid            synthesis; (x) an inhibitor of cholesterol synthesis;            and (xi) any combination of the membrane penetration            enhancing agents recited in (i)-(x);    -   (h) a modulatory agent of epithelial junction physiology;    -   (i) a vasodilator agent;    -   (j) a selective transport-enhancing agent; and    -   (k) a stabilizing delivery vehicle, carrier, mucoadhesive,        support or complex-forming species with which the compound is        effectively combined, associated, contained, encapsulated or        bound resulting in stabilization of the compound for enhanced        nasal mucosal delivery, wherein the formulation of the compound        with the intranasal delivery-enhancing agents provides for        increased bioavailability of the compound in a blood plasma of a        subject.

Additional mucosal delivery-enhancing agents include, for example,citric acid, sodium citrate, propylene glycol, glycerin, ascorbic acid(e.g., L-ascorbic acid), sodium metabisulfite,ethylenediaminetetraacetic acid (EDTA) disodium, benzalkonium chloride,sodium hydroxide, and mixtures thereof. For example, EDTA or its salts(e.g., sodium or potassium) are employed in amounts ranging from about0.01% to 2% by weight of the composition containing alkyl saccharidepreservative.

Therapeutic agents or drugs of the present invention can be peptides orproteins, medically or diagnostically useful, of small to medium size,e.g. up to about 15 kD, 30 kD, 50 kD, 75 kD, etc., or a protein havingbetween about 1-300 amino acids or more. The methods of the inventionalso anticipate the use of small molecules, for example, an organiccompound that has a molecular weight of less than 3 kD, or less than 1.5kD.

The mechanisms of improved drug absorption according to the inventionare generally applicable and should apply to all such peptides orprotein, although the degree to which their absorption is improved mayvary according to the molecular weight (MW) and the physicochemicalproperties of the peptide or protein, and the particular enhancer used.Examples of peptides or protein include vasopressin, vasopressinpolypeptide analogs, desmopressin, glucagon, corticotropin (ACTH),gonadotropin, calcitonin, C-peptide of insulin, parathyroid hormone(PTH), growth hormone (HG), human growth hormone (hGH), growth hormonereleasing hormone (GHRH), oxytocin, corticotropin releasing hormone(CRH), somatostatin or somatostatin polypeptide analogs, gonadotropinagonist or gonadotrophin agonist polypeptide analogs, human atrialnatriuretic peptide (ANP), human thyroxine releasing hormone (TRH),follicle stimulating hormone (FSH), and prolactin.

One preferred composition of the invention is the peptide drug isExenatide (or exendin-4) and an alkyl glycoside. Exenatide is asynthetic version of exendin-4, and has been used in clinical trials byAmylin™ Pharmaceuticals. Exendin-4 is a low molecular weight peptidethat is the first of a new class of therapeutic medications known asincretin mimetic agents or hormones. Incretin hormones are any ofvarious gastrointestinal (GI) hormones and factors that act as potentstimulators of insulin secretion, e.g. as gastric inhibitory polypeptide(GIP), glucagon-like peptide-1 (GLP-1), or Exenatide, or exendin-4, orequivalents thereof.

Exendin-4 is a naturally occurring 39-amino acid peptide isolated fromsalivary secretions of the Gila Monster Lizard. Eng et al., “Isolationand characterization of exendin-4, an exendin-3 analogue, from Helodermasuspectum venom. Further evidence for an exendin receptor on dispersedacini from guinea pig pancreas,” J. Biol. Chem. 267(15):7402-7405(1992). Exenatide exhibits similar glucose lowering actions to glucagonslike peptide, or GLP-1. Exenatide is being investigated for itspotential to address important unmet medical needs of many people withtype 2 diabetes. Clinical trials suggest that Exenatide treatmentdecreases blood glucose toward target levels and is associated withweight loss. The effects on glucose control observed with Exenatidetreatment are likely due to several actions that are similar to those ofthe naturally occurring incretin hormone GLP-1 (see Example 7). Theseactions include stimulating the body's ability to produce insulin inresponse to elevated levels of blood glucose, inhibiting the release ofglucagon following meals and slowing the rate at which nutrients areabsorbed into the bloodstream. In animal studies Exenatideadministration resulted in preservation and formation of new beta cells,the insulin-producing cells in the pancreas, which fail as type 2diabetes progresses.

Use of Exenatide, incretin mimetic agents or equivalents thereof can beused to treat various forms of diabetes including but not limited tobrittle diabetes, chemical diabetes or impaired glucose tolerance,gestational diabetes, diabetes insipidus, diabetes insipidus central,diabetes insipidus nephrogenic, diabetes insipidus pituitary, latentdiabetes, lipatrophic diabetes, maturity-onset diabetes of youth (MODY),diabetes mellitus (DM), diabetes mellitus adult-onset (type 2 DM),diabetes mellitus insulin-dependent (IDDM, or type 1 DM), diabetesmellitus non-insulin dependent (NIDDM), diabetes mellitus juvenile orjuvenile-onset, diabetes mellitus ketosis-prone, diabetes mellitusketosis-resistant, diabetes mellitus malnutrition-related (MRDM),diabetes mellitus tropical or tropical pancreatic, diabetes mellitus,preclinical diabetes, or diabetes induced by various drugs e.g. thiazidediabetes, steroid diabetes, or various diabetes animal model includingbut not limited to alloxan diabetes and puncture diabetes.

In another aspect, therapeutic compositions of the invention are used totreat obesity. Obesity is a common problem in both adults andadolescents. For example, PYY3-36 (or AC162352) is a hormone that playsa critical role in decreasing appetites. The gut hormone fragmentpeptide PYY3-36 (PYY) reduces appetite and food intake when infused intosubjects of normal weight. Similar to the adipocyte hormone, leptin, PYYreduces food intake by modulating appetite circuits in the hypothalamus.However, in obese patients there is a resistance to the action ofleptin, thereby limiting leptin's therapeutic effectiveness. Still otherstudies show that PYY reduces food intake. Injection of PYY revealedthat they eat on average 30% less than usual, resulting in weight loss.Hence, PYY 3-36 has potential as a treatment for obesity. Amylin™Pharmaceuticals submitted an Investigational New Drug application forPYY 3-36 in 2003.

Compounds whose absorption can be increased by the method of thisinvention include any compounds now known or later discovered, inparticular drugs, or therapeutic compounds, molecules or agents that aredifficult to administer by other methods, for example, drugs that aredegraded in the gastrointestinal (GI) tract or that are not absorbedwell from the GI tract, or drugs that subjects could administer tothemselves more readily via the ocular, nasal, nasolacrimal, inhalationor pulmonary, oral cavity (sublingual or Buccal cell), or CSF deliveryroute than by traditional self-administration methods such as injection.Some specific examples include peptides, polypeptides, proteins andother macromolecules, for example, peptide hormones, such as insulin andcalcitonin, enkephalins, glucagon and hypoglycemic agents such astolbutamide and glyburide, and agents which are poorly absorbed byenteral routes, such as griseofulvin, an antifungal agent. Othercompounds include, for example, nicotine, interferon (e.g., alpha, beta,gamma), PYY, GLP-1, synthetic exendin-4 (Exenatide), parathyroid hormone(PTH), and human growth hormone or other low molecular weight peptidesand proteins.

Further, the therapeutic compositions of the invention also contemplatenon-peptide drugs or therapeutic agents. For example, in U.S. Pat. No.5,552,534, non-peptide compounds are disclosed which mimic or inhibitthe chemical and/or biological activity of a variety of peptides. Suchcompounds can be produced by appending to certain core species, such asthe tetrahydropyranyl ring, chemical functional groups which cause thecompounds to be at least partially cross-reactive with the peptide. Aswill be recognized, compounds which mimic or inhibit peptides are tovarying degrees cross-reactivity therewith. Other techniques forpreparing peptidomimetics are disclosed in U.S. Pat. Nos. 5,550,251 and5,288,707. The above U.S. patents are incorporated by reference in theirentirety.

The method of the invention can also include the administration, alongwith the alkyl glycoside and a protein or peptide, a protease orpeptidase inhibitor, such as aprotinin, bestatin, alpha₁, proteinaseinhibitor, soybean trypsin inhibitor, recombinant secretory leucocyteprotease inhibitor, captopril and other angiotensin converting enzyme(ACE) inhibitors and thiorphan, to aid the protein or peptide inreaching its site of activity in the body in an active state (i.e., withdegradation minimal enough that the protein is still able to functionproperly). The protease or peptidase inhibitor can be mixed with thealkyl glycoside and drug and then administered, or it can beadministered separately, either prior to or after administration of theglycoside or drug.

The invention also provides a method of lowering blood glucose level ina subject comprising administering a blood glucose-reducing amount of acomposition comprising insulin and an absorption increasing amount of asuitable nontoxic, nonionic alkyl glycoside having a hydrophobic alkylgroup joined by a linkage to a hydrophilic saccharide, therebyincreasing the absorption of insulin and lowering the level of bloodglucose. A “blood glucose-reducing amount” of such a composition is thatamount capable of producing the effect of reducing blood glucose levels,as taught herein. Preferred is an amount that decreases blood glucose tonormoglycemic or near normoglycemic range. Also preferred is an amountthat causes a sustained reduction in blood glucose levels. Even morepreferred is an amount sufficient to treat diabetes, including diabetesmellitus (DM) by lowering blood glucose level. Thus, the instant methodcan be used to treat diabetes mellitus. Preferred alkyl glycosides arethe same as those described above and exemplified in the Examples.

Also provided is a method of raising blood glucose level in a subject byadministering a blood glucose-raising amount comprising glucagons and atleast one alkyl glycoside and/or saccharide alkyl ester. When thecomposition includes insulin, it can be used to cause the known effectof insulin in the bloodstream, i.e., lower the blood glucose levels in asubject. Such administration can be used to treat diabetes mellitus, orrelated diseases. A “blood glucose-raising amount” of glucagon in such acomposition is that amount capable of producing the effect of raisingblood glucose levels. A preferred amount is that which increases bloodglucose to normoglycemic or near-normoglycemic range. Another preferableamount is that which causes a sustained rising of blood glucose levels.Even more preferred, is that amount which is sufficient to treathypoglycemia by raising blood glucose level. Thus, this method can beused to treat hypoglycemia. Preferred alkyl glycosides are the same asthose described above and exemplified in the Examples.

Similarly, when this composition includes glucagon, it can be used tocause the known effect of glucagon in the bloodstream, i.e., to raisethe blood glucose levels in a subject. Such administration can thereforebe used to treat hypoglycemia, including hypoglycemic crisis.

The invention also provides methods for ameliorating neurologicaldisorders which comprises administering a therapeutic agent to thecerebral spinal fluid (CSF). The term “neurological disorder” denotesany disorder which is present in the brain, spinal column, and relatedtissues, such as the meninges, which are responsive to an appropriatetherapeutic agent. The surprising ability of therapeutic agents of thepresent invention to ameliorate the neurological disorder is due to thepresentation of the therapeutic agent to persist in thecerebro-ventricular space. The ability of the method of the invention toallow the therapeutic agent to persist in the region of the neurologicaldisorder provides a particularly effective means for treating thosedisorders.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject in need of treatment maybe varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy. Generally, however, dosage will approximate thatwhich is typical for known methods of administration of the specificcompound. For example, for intranasal administration of insulin, anapproximate dosage would be about 0.5 unit/kg regular porcine insulin(Moses et al.). Dosage for compounds affecting blood glucose levelsoptimally would be that required to achieve proper glucose levels, forexample, to a normal range of about 5-6.7 mM. Additionally, anappropriate amount may be determined by one of ordinary skill in the artusing only routine testing given the teachings herein (see Examples).

Furthermore, the compositions of the invention can be administered in aformat selected from the group consisting of a drop, a spray, an aerosoland a sustained release format. The spray and the aerosol can beachieved through use of the appropriate dispenser. The sustained releaseformat can be an ocular insert, erodible microparticulates, swellingmucoadhesive particulates, pH sensitive microparticulates,nanoparticles/latex systems, ion-exchange resins and other polymericgels and implants (Ocusert, Alza Corp., California; Joshi, A., S. Pingand K. J. Himmelstein, Patent Application WO 91/19481). These systemsmaintain prolonged drug contact with the absorptive surface preventingwashout and nonproductive drug loss.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. The following examples are intended to illustrate but notlimit the invention.

EXAMPLE 1 Alkyl Glycoside and/or Sucrose Ester Formulations do not CauseMucosa Irritation or Disruption

The nasal mucosa is highly vascularized and hence optimal for high drugpermeation. Moreover, absorption of drug(s) through the nasal mucosa isavailable to the central nervous system (CNS). Although localapplication of drugs is desirable, a challenge for this method ofadministration is mucosal irritancy.

A formulation consisting of an alkyl glycoside (0.125% TDM) in acommercial over-the-counter (OTC) nasal saline was administered in vivoto human nasal epithelium over a period of over one month. The 0.125%TDM formulation is compared to the control, namely the same commercial(OTC) nasal saline, over the same period of time. Results show thatduring and after 33 days of daily TDM administration (i.e., the durationof the study), there is no observable irritation of the nasal mucosa(data not shown). Thus, compositions of the invention are non-toxic andnon-irritable providing repeated and long-term intranasaladministration, which is beneficial for those patients with chronic andongoing disease(s).

A similar test was performed using sucrose dodecanoate, a sucrose ester.Sucrose dodecanoate is administered in vivo to human nasal epitheliumand during and after 47 days (i.e., the duration of the study), noobservable irritation was detected (data not shown). Thus, these resultsshow that alkyl glycosides and sucrose esters of the invention arenon-toxic and do not cause mucosa irritation when administered dailyover a long period of time.

EXAMPLE 2 Alkyl Glycoside and/or Sucrose Ester Compositions StabilizeDrugs by Increasing Drug Bioavailability and Reducing DrugBioavailability Variance

Stability of the alkyl glycoside depends, in part, on the number ofcarbon atoms or length of the alkyl chain and other long alkyl chains,with tetradecylmaltoside (TDM) having the greatest effect; but otherhighly branched alkyl chains including DDM also have stabilizingeffects. In contrast to Hovgaard-1, which described the preference for ahigh alkyl glycoside to drug ratio, the instant invention shows thatthis ratio is much lower. For example, alkyl glycosides in the range ofabout 0.01% to about 6% by weight result in good stabilization of thedrug; whereas Hovgaard-1 shows stabilization is only achieved at muchhigher ratios of alkyl glycosides to drug (10:1 and 16:1). Even moreinteresting, alkyl glycosides of the invention in the range of about0.01% to about 6% have increased bioavailability (see FIG. 1). This isin sharp contrast to Hovgaard-2, which showed relatively lowbioavailability (0.5-1%) at the high alkyl glycoside ratios (10:1 and16:1).

FIG. 1 is a graph comparing the bioavailability of the drug MIACALCIN®(salmon calcitonin from Novartis) with and without alkyl glycoside(TDM). MIACALCIN® is a nasal spray and administered directly onto thenasal epithelium or nasal mucosa. FIG. 1 shows that MIACALCIN® minusalkyl glycoside has very low bioavailability levels in humans(MIACALCIN® product specification insert), as compared to the MIACALCIN®with alkyl glycoside as administered to rats. More specifically,intranasal delivery of MIACALCIN® with 0.125% and 0.250% alkyl glycoside(TDM) resulted in about 43% to about 90% bioavailability, respectively.The bioavailability of intranasal administration of MIACALCIN® withoutalkyl glycoside is only about 3% in humans, and was undetectable inrats, suggesting that the rat is a stringent model for estimatingintranasal drug absorption in humans. Thus, the alkyl glycoside of theinvention enhances absorption and increases bioavailability of the drug.

Furthermore, besides increasing the bioavailability of the drug, thealkyl glycoside compositions of the invention effectively decrease thebioavailability variance of the drug. FIG. 1 shows that administrationof MIACALCIN® with alkyl glycoside (0.125% or 0.25%) intranasally has abioavailability variance of +/−8%, whereas the bioavailability variancewithout alkyl glycoside is 0.3% to 30%, or a two orders of magnitudechange. The increase in bioavailability and the decrease in thebioavailability variance ensures patient-to-patient variability is alsoreduced. The results as shown in FIG. 1 are administered intranasally,however, similar results are expected for oral, buccal, vaginal, rectal,etc. delivery and at different alkyl glycoside concentrations.

Thus, contrary to the art, the alkyl glycoside compositions of theinvention, in the range of about 0.01% to about 6% result in increasedbioavailability and reduced bioavailability variance. This has nototherwise been reported.

EXAMPLE 3 Ocular Administration of Alkyl Saccharides Plus InsulinProduces Hypoglycemic Effects In Vivo

Normal rats were anesthetized with a mixture of xylazine/ketamine toelevate their blood glucose levels. The elevated levels of D-glucosethat occur in response to anesthesia provide an optimal system tomeasure the systemic hypoglycemic action of drug administration, e.g.insulin-containing eye drops. This animal model mimics the hyperglycemicstate seen in diabetic animals and humans. In the experimental animalgroup, anesthetized rats are given eye drops containing insulin. Bloodglucose levels from the experimental group are compared to anesthetizedanimals which received eye drops without insulin. The change in bloodglucose levels and the differential systemic responses reflects theeffect of insulin absorbed via the route of administration, e.g. ocularroute.

Adult male Sprague-Dawley rats (250-350 g) were fed ad libitum, andexperiments were conducted between 10:00 a.m. and 3:00 p.m. Rats wereanesthetized with a mixture of xylazine (7.5 mg/kg) and ketamine (50mg/kg) given intraperitoneally (IP) and allowed to stabilize for 50-90min before the administration of eye drops. Anesthesia of a normal ratwith xylazine/ketamine produces an elevation in blood glucose valueswhich provides an optimal state to determine the systemic hypoglycemicaction of insulin-containing eye drops. Blood D-glucose values weremeasured by collecting a drop of blood from the tail vein at 5-10 minintervals throughout the experiment and applying the blood to glucometerstrips (Chemstrip bG) according to directions provided with theinstrument (Accu-Chek II, Boehringer Mannheim Diagnostics; Indianapolis,Ind.). Blood D-glucose values ranged from 200 to 400 mg/dl inanesthetized nondiabetic rats.

At time 0, after a 50-90 min stabilization period, rats were given 20 μlof eye drops composed of phosphate-buffered saline (PBS) with or without0.2% regular porcine insulin and 0.125%-0.5% of the absorption enhancingalkyl glycoside (e.g. TDM) to be tested. Eye drops were instilled attime 0 using a plastic disposable pipette tip with the eyes held open,and the rat was kept in a horizontal position on a warming pad (37° C.)throughout the protocol. The rats were given additional anesthesia ifthey showed signs of awakening. Rats received in each eye 20 μl of0.125-0.5% absorption enhancer in phosphate buffered saline, pH 7.4 with(experimental) or without (control) 0.2% (50 U/ml) regular porcineinsulin (Squibb-Novo, Inc.) for a total of 2 U per animal.Octyl-β-D-maltoside, decyl-β-D-maltoside, dodecyl-μ-D-maltoside,tridecyl-β-D-maltoside and tetradecyl-β-D-maltoside were obtained fromAnatrace, Inc. (Maumee, Ohio). Hexylglucopyranoside,heptylglucopyranoside, nonylglucopyranoside, decylsucrose anddodecylsucrose were obtained from Calbiochem, Inc. (San Diego, Calif.);Saponin, BL-9 and Brij 78 were obtained from Sigma Chemical Co. (St.Louis, Mo.).

The D-glucose levels in the blood remained elevated when the animalsreceived eye drops containing: 1) saline only; 2) 0.2% regular porcineinsulin in saline only; or 3) absorption enhancer only. However, whenrats received eye drops containing 0.2% regular porcine insulin andseveral alkylmaltoside or alkylsucrose compounds, a pronounced decreasein blood D-glucose values occurred and was maintained for up to twohours. Insulin administered ocularly with 0.5% dodecyl-β-D-maltoside(see Table I) or 0.5% decyl-β-D-maltoside (see Table III) results in aprompt and sustained fall in blood glucose levels which are maintainedin the normoglycemic (80-120 mg/dl) or near-normoglycemic (120-160mg/dl) range for the two hour duration of the experiment. Hence, atleast two alkylmaltosides are effective in achieving sufficientabsorption of insulin delivered via the ocular route to produce a promptand sustained fall in blood glucose levels in experimentallyhyperglycemic animals. The surfactant compositions of the invention aretherefore useful to achieve systemic absorption of insulin and otherpeptides/proteins, e.g., glucagon and macromolecular drugs and heparindelivered via the ocular route in the form of eye drops.

Several other alkylmaltosides are also effective as absorption enhancersfor ocular administration of insulin including 0.5% tridecylmaltoside(see Table III) and 0.125% (Table II) and 0.5% tetradecyl maltoside.These studies show that alkylmaltosides with the longer alkyl chains (ornumber of carbon atoms), e.g., dodecyl-, tridecyl- andtetradecyl-β-D-maltosides, are more effective. The increase in thenumber of carbon atoms also contributes to the greaterhydrophobic/hydrophilic structural balance and absorption enhancingeffect. The shorter alkyl chains (fewer carbon atoms) e.g.,decylmaltoside, or no, e.g., octylmaltoside, produce less absorptionenhancing activity. It is noted that the most effective alkylmaltosidesproduce effects comparable to or greater than those seen with otherabsorption enhancers such as saponin, and with the added advantage thatthey can be metabolized to nontoxic products following systemicabsorption.

The effects of the alkylmaltosides as absorption enhancers aredose-dependent, as can be seen by examining the effects of differentconcentrations ranging from 0.125-0.5% in producing a hypoglycemiceffect when combined with insulin. Whereas, 0.5% and 0.375%dodecylmaltoside appear equally effective in achieving systemicabsorption of insulin and reduction of blood glucose levels, 0.25% has asmaller and more transient effect and 0.125% is ineffective (Table I).Similarly, tridecylmaltoside also shows a dose-dependent effect inlowering blood glucose concentrations when combined with insulin, butthe effect achieved with even 0.25% of the absorption enhance issustained for the two hour time course of the experiment. Thus,dose-dependent effects of the alkylmaltosides suggest that they achieveenhancement of protein absorption via the ocular route in a gradedfashion proportional to the concentration of the agent.

TABLE I Effect of Eye Drops Containing Insulin Plus VariousConcentrations of Dodecyl Maltoside on Blood Glucose Values (in mg/dl)in Rat Dodecyl Maltoside Concentration 0.125% 0.25% 0.375% 0.50% Time(min) Blood Glucose Concentrations (mg/dl) −20 305 ± 60 271 ± 38 305 ±51 375 ± 9 −10 333 ± 58 295 ± 32 308 ± 27 366 ± 12 0 338 ± 67 323 ± 62309 ± 32 379 ± 4 30 349 ± 64 250 ± 48 212 ± 18 297 ± 18 60 318 ± 38 168± 22 134 ± 4 188 ± 25 90 325 ± 57 188 ± 55 125 ± 12 141 ± 13 120 342 ±78 206 ± 63 119 ± 19 123 ± 5

The absorption enhancing effects of the alkyl saccharides were notconfined to the alkylmaltosides alone since dodecylsucrose (0.125%,0.25%, 0.375%) also shows a dose-dependent effect in producing ocularabsorption of insulin and reduction in blood glucose levels. This effectis observed even at 0.125% alkyl saccharide (from 335 mg/dl.+−0.26 mg/dlat time 0 min. to 150 mg/dl+−0.44 mg/dl at time 120 min.). 0.5%decylsucrose was also effective in reducing blood glucose levels, but asshown for the alkylmaltosides, a reduction in the length of the alkylchain, and hence the hydrophobic properties of the molecule, appears toreduce the potency of the alkylsucrose compounds. However, a significantand sustained reduction in blood glucose levels is achieved with 0.5%decylsucrose (from 313 mg/dl.+−0.15 mg/dl at time 0 min. to 164mg/dl+−0.51 mg/dl at time 120 min.). The absorption enhancing abilitiesof alkyl saccharides with two distinct disaccharide moieties suggeststhat it is the physicochemical properties of the compounds which arecrucial to their activity and that other alkyl saccharides, e.g.,dodecyllactose, have the right balance of properties to be equally ormore effective as absorption enhancers while retaining the metabolic andnontoxic properties of the alkylsaccharide enhancing agents. These alkylsaccharides are anticipated by the invention.

Studies with alkylglucosides were also conducted; 0.5% hexylglucosideand 0.5% heptylglucoside were ineffective at promoting insulinabsorption from the eye, but 0.5% nonylglucoside effectively stimulatedinsulin absorption and reduced blood glucose levels (from 297 mg/dl to150 mg/dl). This result once further supports that the alkyl chainlength, as well as the carbohydrate moiety, play critical roles ineffectively enhancing insulin absorption.

It should be noted that no damaging effects (i.e. non-irritants) to theocular surface were observed with any of the alkylmaltoside oralkylsucrose agents employed in these studies. Furthermore, the promptand sustained hypoglycemic effects produced by these agents incombination with insulin suggest that these absorption enhancers do notadversely affect the biological activity of the hormone, in keeping withtheir nondenaturing, mild surfactant properties.

Thus, therapeutic compositions on the invention consisting of at leastan alkyl glycoside and a drug are stable and the alkyl glycosidesenhance the absorption of the drug.

EXAMPLE 4 Ocular and Intranasal Administration of TDM Plus GlucagonProduces Hypoglycemic Effects In Vivo

Since previous Examples showed that administration via eye drops of anabsorption enhancer with drug e.g. insulin results in significantabsorption of the drug via the nasolacrimal drainage system,therapeutically effective administration of insulin withalkylmaltosides, alkylsucrose and like agents by intranasaladministration is tested herein.

Tetradecylmaltoside (TDM) in combination with insulin also produced adrop in blood D-glucose levels when administered in the form of a dropintranasally as well as via a drop by the ocular route. Eye dropscontaining 0.2% regular porcine insulin with 0.125% tetradecylmaltosideare administered to rats as previously described. The administration ofthe composition produces a prompt and prominent drop in blood glucoselevels. The drop in blood glucose levels decrease even more byadministration of a nose drop containing the same concentration ofinsulin with 0.5% tetradecylmaltoside (Table II). Thus, intranasaldelivery and administration of the alkyl saccharide with drug results inlowering of blood glucose levels.

TABLE II Effect of Insulin Eye Drops, Containing 0.125% TetradecylMaltoside and Nose Drops Containing 0.5% Tetradecyl Maltoside on BloodGlucose Values in Rats Time (min) Blood Glucose (mg/dl) −20 319 −10 311Eye drops added 0 322 15 335 30 276 45 221 60 212 75 167 90 174 105 167120 208 Nose Drops Added 135 129 150 74 165 76 180 68

EXAMPLE 5 Ocular Administration of Alkyl Saccharides Plus InsulinProduces Hyperglycemic Effects In Vivo

Previous studies demonstrated that insulin absorption from the eye isstimulated by saponin, BL-9 and Brij-78. BL-9 and Brij-78 areineffective at stimulating the absorption of glucagon from the eye,whereas saponin is effective. Glucagon absorption from the eye wasmeasured in rats given eye drops containing various surfactants plusglucagon (30 μg) (Eli Lilly, Indianapolis, Ind.) by monitoring anelevation in blood D-glucose levels. In these experiments, rats wereanesthetized with sodium pentobarbital rather than xylazine/ketamin.This modification of the procedure resulted in basal blood glucoselevels in the normoglycemic range and made it possible to readilymonitor the hyperglycemic action of any glucagon absorbed from the eye.

Paired animals that receive eye drops containing the surfactant alone,or glucagon alone, were compared to animals receiving eye drops with thesurfactant plus glucagon. When eyedrops containing 0.5% saponin plusglucagon are administered to rats, the level of D-glucose in blood risessignificantly, but no such effect is observed with eye drops containing0.5% BL-9 or 0.5% Brij-78 plus glucagon. Interestingly, when eye dropscontaining dodecylsucrose, decylmaltose or tridecylmaltose plus glucagonare administered to rats which were previously treated with eye dropscontaining these surfactant agents plus insulin, the glucagon isabsorbed and blood D-glucose values increase significantly (Table III).This result confirms that ocular administration of certainalkylsaccharides can enhance the absorption of drugs, including glucagonand insulin. Moreover, it is now possible to treat for a hypoglycemiccrisis using a formulation with at least an alkyl saccharide of theinvention.

TABLE III Effect of Eye Drops Containing Insulin or Glucagon and 0.5%Decyl Maltoside, 0.5% Dodecyl Sucrose, or 0.5% Tridecyl Maltoside onBlood Glucose Values in Rats Surfactant Agent Dodecyl Sucrose DecylMaltoside Tridecyl Maltoside Time (min) Blood Glucose Concentration(mg/dl) −20 266 249 255 −10 305 287 307 Insulin Eye Drops Added 0 351337 323 10 347 304 309 20 252 292 217 30 161 221 131 40 120 164 100 50105 138 87 60 114 114 107 70 113 104 115 80 104 110 79 90 86 120 85 100113 92 76 110 107 81 74 120 112 87 75 Glucagon Eye Drops Added 130 11195 82 140 143 99 121 150 202 132 148 160 247 157 173 170 242 171 162 180234 180 162 190 211 189 156

EXAMPLE 6 Intranasal Administration of 0.25% TDM Plus Insulin DecreasesBlood Glucose Levels In Vivo

Intranasal administration of drugs or agents are possible in animalmodels e.g. mice and rats, although the nasal opening in is very small.In the experiments and results described herein, an anesthesia-inducedhyperglycemia model was used (described in Examples above).Hyperglycemic animals were induced by an intraperitoneal (IP) injectioncontaining xylazine-ketamine and blood glucose levels were monitoredover a period of time. Immediately after the xylazine-ketamineinjection, there was an increase in the blood glucose levels as shown inFIG. 2 (closed dark circles), and blood glucose levels were about 450mg/dl. The increase in blood glucose levels was attributed to theinhibition of pancreatic insulin secretion. Blood glucose levels peak toabout 482 mg/dl by 30 minutes after the xylazine-ketamine injection(FIG. 2). Then, at approximately 33 minutes after the xylazine-ketamineinjection, 6 μL of insulin (Humalog) in 0.25% tetradecylmaltoside (TDM;or Intravail A) was administered intranasally using a long thinmicropipette tip, and blood glucose levels were monitored at about 15minute intervals. After administration of the 0.25% TDM/insulincomposition, there was a rapid decrease in blood glucose levels,reaching a low of about 80 mg/dl at about the 60 minute time point, orabout 30 minutes after the insulin administration (FIG. 2). At about the75 minute time point, blood glucose levels gradually returned to thebaseline level in a normoglycemic mouse, or about 80-100 mg/dl.

The results above were compared with animals treated with insulin alone(same dosage), minus 0.25% TDM (FIG. 2, open circles). The insulin onlytreatment showed blood glucose levels do not start to decline until atabout the 120 minute time mark, or about 110 minutes after the insulinadministration. Further, the blood glucose levels observed in animalstreated with insulin alone never return to normoglycemic levels, as wasobserved in those animals receiving insulin plus 0.25% TDM (FIG. 2).

Thus, these results again demonstrate that compositions of the inventionconsisting of certain alkyl glycosides or alkyl saccharides plus a drug,e.g. insulin, effectively lower blood glucose levels, and that theseeffects are measurable shortly after administration of the drug.

EXAMPLE 7 Intranasal Administration of 0.25% TDM (Intravail A)+Exendin-4Decreases Blood Glucose Levels In Vivo

The ob/ob mouse model was utilized for the studies described herein.Friedman, J. M., Nature 404, 632-634 (2000). All animals received anintraperitoneal (IP) injection of a bolus of 2 g/kg glucose for purposesof determining glucose tolerance. At time 0 the experimental animalswere given about 100 micrograms/kg of exendin-4/0.25% TDM (exendin-4from American Peptide) either as 10 μl of nasal drops (FIG. 3; closedtriangles), or by IP injection (FIG. 3; closed circles), or by and IPinjection of saline alone (no drug, no TDM; FIG. 3; open circles).Control animals were previously performed and received no drugs. Theresults of this study are shown in FIG. 3.

FIG. 3 shows that glucose tolerance of the animals were different sinceblood glucose levels vary at time 0 when the animals received theglucose bolus. Regardless, of the glucose tolerance level at time 0,immediately after injection of the glucose bolus, blood glucose levelsincreased in all three animals. The blood glucose level of the animalreceiving the IP injection of saline alone does not decrease as rapidlyas the experimental animals receiving the drug. Moreover, the animalreceiving the IP injection of saline alone never reached a normoglycemiclevel (FIG. 3, open circles). In contrast, the experimental animals,after administration of nasal drops of exendin-4/TDM, or IP injection ofexendin-4/TDM, showed a rapid and immediate decrease in blood glucoselevels.

Also exendin-4 administered about 15-30 minutes ahead of the glucosebolus (before time 0 in FIG. 3; data not shown) produced an even morepronounced lowering of blood glucose effect, because the absorption ofthe hormone takes a certain amount of time to be absorbed and to beactive. Thus, exendin-4 (or Exenatide) which is currently in humanclinical trials, when combined with alkyl glycosides of the invention,effectively treats a hyperglycemic condition by lowering the bloodglucose levels of the hyperglycemic subject.

EXAMPLE 8 Alkyglycosides have Antibacterial Activity by ReducingBacterial Log Growth

The cultures of Candida albicans (ATCC No. 10231), Aspergillus niger(ATCC No. 16404), Escherichia coli (ATCC No, 8739), Pseudomonasaeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538)were obtained from American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209. The viable microorganisms used inthe invention were not more than five passages removed from the originalATCC culture. As described herein, one passage is defined as thetransfer of organisms from an established culture to fresh medium andall transfers are counted.

Cultures received from the ATCC are resuscitated according to thedirections provided by the ATTC. Cells grown in broth were pelleted bycentrifugation, resuspended in 1/20th the volume of fresh maintenancebroth, and combined with an equal volume of 20% (v/v in water) sterileglycerol. Cells grown on agar were scraped from the surface into themaintenance broth also containing 10% glycerol broth. Small aliquots ofthe suspension were dispensed into sterile vials and the vials werestored in liquid nitrogen or in a mechanical freezer at a temperature nohigher than about −50° C. When a fresh seed-stock vial was required, itwas removed and used to inoculate a series of working stock cultures.These working stock cultures were then used periodically (each day inthe case of bacteria and yeast) to start the inoculum culture.

All media described herein should be tested for growth promotion usingthe microorganisms indicated above under Test Organisms.

To determine whether the alkyl saccharides of the invention inhibitgrowth or have antibacterial activity, the surface of a suitable volumeof solid agar medium was inoculated from a fresh revived stock cultureof each of the specified microorganisms. The culture conditions for theinoculum culture is substantially as described in Table IV. For example,suitable media can include but is not limited to, Soybean-Casein Digestor Sabouraud Dextrose Agar Medium. The bacterial and C. albicanscultures was harvested using sterile saline TS, by washing the surfacegrowth, collecting it in a suitable vessel, and adding sufficientsterile saline TS to obtain a microbial count of about 1×10⁸colony-forming units (cfu) per mL. To harvest the cells of A. niger, asterile saline TS containing 0.05% of polysorbate 80 was used, and thenadding sufficient sterile saline TS to obtain a count of about 1×10⁸ cfuper mL.

Alternatively, the stock culture organisms may be grown in any suitableliquid medium (e.g., Soybean-Casein Digest Broth or Sabouraud DextroseBroth) and the cells harvested by centrifugation, and washed andresuspended in sterile saline TS to obtain a microbial count of about1×10⁸ cfu per mL. The estimate of inoculum concentration was determinedby turbidimetric measurements for the challenge microorganisms. Thesuspension should be refrigerated if it is not used within 2 hours. Toconfirm the initial cfu per mL estimate, the number of cfu per mL ineach suspension was determined using the conditions of media andmicrobial recovery incubation times listed in Table IV (e.g., from about3 to about 7 days). This value serves to calibrate the size of inoculumused in the test. The bacterial and yeast suspensions were used within24 hours of harvest; whereas the fungal preparation can be stored underrefrigeration for up to 7 days.

TABLE IV Culture Conditions for Inoculum Preparation MicrobialIncubation Inoculum Recovery Organism Suitable Medium TemperatureIncubation Time Incubation Time Escherichia coli Soybean-Casein Digest32.5 ± 2.5 18 to 24 3 to 5 days (ATCC No. 8739) Broth; hoursSoybean-Casein Digest Agar Staphylococcus Soybean-Casein Digest 32.5 ±2.5 18 to 24 3 to 5 days aureus Broth; hours (ATCC No. 6538)Soybean-Casein Digest Agar Candida albicans Sabouraud Dextrose Agar;22.5 ± 2.5 44 to 52 3 to 5 days (ATCC No. 10231) Sabouraud DextroseBroth hours Aspergillus niger Sabouraud Dextrose Agar; 22.5 ± 2.5 6 to10 days 3 to 7 days (ATCC No. 16404) Sabouraud Dextrose Broth

To determine which alkylglycoside formulations have antibacterialactivity, the formulations were prepared in phosphate buffered saline(PBS) at pH 7. As a source of nutrition, either 1.5 mg/mL bovine serumalbumin (BSA; see Tables V and VI) or 1 mg/mL of PYY was added (seeTable VII) to the medium. BSA (CAS Number: 9048-46-8) was obtained fromSigma-Aldrich, St. Louis, Mo., USA,n-dodecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside andn-tetradecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside were obtainedfrom Anatrace Inc., Maumee, Ohio, USA, and PYY was obtained from BachemCalifornia Inc., Torrance, Calif., USA.

Antibacterial activity of the alkylglycosides were conducted in foursterile, capped bacteriological containers of suitable size into which asufficient volume of alkylglycoside solution had been transferred. Eachcontainer was inoculated with one of the prepared and standardizedinoculums and mixed. The volume of the suspension inoculum was betweenabout 0.5% and about 1.0% of the volume of the alkylglycoside solution.The concentrations of test microorganisms added to the alkylglycosidesolution was such that the final concentrations of the test preparationafter inoculation was between about 1×10⁵ and 1×10⁶ cfu per mL ofalkylglycoside solution. To determine the level of inhibition of growth,or reduction of growth based on a logarithmic scale, the initialconcentration of viable microorganisms in each test preparation wasestimated based on the concentration of microorganisms in each of thestandardized inoculum as determined by the plate-count method. Theinoculated containers were then incubated at about 22.5° C.±2.5. Thegrowth or non-growth of the microorganisms in each culture/containerwere again determined at day 14 and day 28. The number of cfu present ineach calculation was determined by the plate-count procedure standard inthe art for the applicable intervals. The change in the orders ofmagnitude of bacterium and/or fungi was then determined by subtractingthe first calculated log 10 values of the concentrations of cfu per mLpresent at the start or beginning (e.g., day 0), from the log 10 valuesof the concentration of cfu per mL for each microorganism at theapplicable test intervals (e.g., day 14 and day 28; see Tables V, VI andVII).

TABLE V Log reduction of microorganisms in cultures containing 0.125%n-Dodecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside StaphlococcusEscherichia Candida Aspergillus aureus Coli albicans niger Day 0 7.3 ×10⁵ 1.2 × 10⁵ 3.2 × 10⁵ 4.8 × 10⁵ (cfu/gm) (cfu/gm) (cfu/gm) (cfu/gm)Day 14 ≧5.2 orders of N.D. 3.0 orders of 0.7 orders of magnitudemagnitude magnitude reduction reduction reduction Day 28 ≧5.2 orders of0.1 orders of ≧5.3 orders of No growth magnitude magnitude magnitudefrom initial reduction reduction reduction count

TABLE VI Log Reductions in cultures containing 0.2%n-Tetradecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside StaphlococcusEscherichia Candida Aspergillus aureus Coli albicans niger Day 0 7.3 ×10⁵ 1.2 × 10⁵ 3.2 × 10³ 4.8 × 10⁵ (cfu/gm) (cfu/gm) (cfu/gm) (cfu/gm)Day 14 ≧5 orders of N.D. 3.0 orders of 0.5 orders of magnitude magnitudemagnitude reduction reduction reduction Day 28 ≧5 orders of No growth≧5.4 orders of No growth magnitude from initial magnitude from initialreduction count reduction count

TABLE VII Log reduction of cultures containing 0.25%n-Dodecyl-4-O-α-D-glucopyranosyl-β-D-glucopyranoside StaphlococcusEscherichia Candida Aspergillus aureus Coli albicans niger Day 0 7.3 ×10⁵ 1.2 × 10⁵ 3.2 × 10⁵ 4.8 × 10⁵ (cfu/gm) (cfu/gm) (cfu/gm) (cfu/gm)Day 14 ≧4.9 orders of ≧5 orders of ≧4.5 orders of 4.7 orders ofmagnitude magnitude magnitude magnitude reduction reduction. reduction.reduction Day 28 ≧4.9 orders of ≧5 orders of ≧4.5 orders of 4.7 ordersof magnitude magnitude magnitude magnitude reduction reduction.reduction. reduction

Determining the antibacterial activity of other alkylglycosides wouldoccur substantially as described herein.

EXAMPLE 9 Administration of Alkyglycosides with AntisenseOligonucleotides to Primates

An approximately 7,000 Dalton antisense oligonucleotide (ASO) with amodified backbone (phosphorothioate oligonucleotide as described in U.S.Pat. No. 7,132,530) mixed with alkylglycosidetetradecyl-beta-D-maltoside (Intravail™), was administered to sixCynomolgus monkeys canulated into the jejunum at a dose of 10 mg/kg. Theanimals were fasted prior to administration. Test agents were dissolvedin PBS buffer and injected through the cannula into the jejunum of eachanimal in a 1.5 mL volume or administered subcutaneously (s.c.) as notedin Table VIII.

TABLE VIII Bioavailability of Antisense Drugs Administered WithTetradecyl-Beta-D-Maltoside Average Bioavailability Test Agents (n = 6)Observations ASO (no tetradecyl- 0% Intestinal villi beta-D-maltoside)(undetectable) completely intact intrajejunal ASO administered 100%Intestinal villi s.c. at 0.5 mg/kg. completely intact 10 mg ASO + 50 18%+/− 7% Intestinal villi mg/kg tetradecyl- completely intactbeta-D-maltoside A5 intrajejunal 10 mg ASO + 50 9% +/− 7% The tops ofsome of the mg/kg sodium intestinal villi were caprate intrajejunalfound to be missing

The protocol involved a 3 way crossover in which each animal had thefirst 3 test agents in Table VIII administered on 3 different dates.There was a 1 week washout period between dosing dates. Two of theanimals were subsequently given a fourth test agent containing 5% sodiumcaprate as an absorption enhancer. Analysis of the blood levels wasconducted using quantitative analysis involving solid phase extractionusing cationic polystyrene nanoparticles.

Solid-phase extractions of the blood samples were first performed.Nanoparticle-oligonucleotide conjugates were formed using a known amountof oligonucleotide added to an aliquot of each sample (200-400 μl) anddiluted with 800 μl of 50 mM Tris.HCl (pH 9) in deionized water. Themixture was briefly vortexted prior to the addition of 200 μl of apolystyrene nanoparticle suspension prepared by surfactant-free emulsionpolymerization using water-soluble cationic initiators to induce apositive surface charge (solid content: approximately 10 mg/ml). Themixture was subsequently vortexed again. After 5-10 min of incubation,the suspension was centrifuged and the supernatant removed. Theparticles were resuspended in 1 ml of a solution of 0.5 M acetic acid indeionized water/ethanol (1:1) and separated from the washing solution bycentrifugation. After the supernatant was removed, the particles wereresuspended in 1 ml of deionized water and separated by anothercentrifugation step. 200 μl of a solution of 150 μM SDS in aqueousammonia (25%)/acetonitrile ( 60/40) was added to thenanoparticle-oligonucleotide conjugates and the releasedoligonucleotides were separated from the carrier by centrifugation. Inorder to exclude contamination of the samples with residual particles,the supernatant was placed in another 1.5-ml tube and centrifuged again.Subsequently, the samples were dried by rotoevaporation orlyophilization and stored at −20° C. until analysis.

Quantitative analysis was performed with capillary gel electrophoresisof the extracted samples. Capillary gel electrophoresis (CGE) wasperformed with a capillary electrophoresis system. An oligonucleotideanalysis kit containing polyvinyl alcohol (PVA) coated capillaries,polymer solution B, and oligonucleotide buffer was obtained. UsingPVA-coated capillaries, analysis was carried out using the manufacturesprotocol.

Using the data obtained from CGE analysis, quantitation ofphosphorothioate oligonucleotides was carried out. The amount ofoligonucleotides in the samples (n_(ON)) was calculated using thefollowing formula:n _(ON) =n _(Std)(ε_(Std)/ε_(ON))((A _(ON) /T _(ON))/(A _(Std) /T_(Std))),where n_(Std) is the amount of standard oligonucleotide added to thesample, ε_(Std) and ε_(ON) are the molar extinction coefficients, andA_(Std)/T_(Std) and A_(ON)/T_(ON) are the corrected peak areas (quotientof peak area and migration time) of the standard and the investigatedcompound, respectively. The quotient of the corrected peak areas of theanalyte and the standard is referred to as the normalized area.

AUC's were calculated from the concentration vs. time cures over a 240minute period. The relative bioavailabilities were determined as theratio of each AUC divided by the AUC for the intravenously administereddrug. Intravail™ (tetradecyl-beta-D-maltoside) excipient providedbioavailability up to 18%. The control showed no detectable absorptionwithout a surfactant excipient. The sodium caprate formulation showed anaverage bioavailability of 9%.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

-   Birkett et al., (1991) “Bioavailability and first pass clearance,”    Austra Prescr 14:14-16.-   Birkett et al., (1990) “How drugs are cleared by the liver,” Austra    Prescr 3:88-89.-   Hovgaard et al., (1996) “Stabilization of insulin by    alkylmaltosides: A spectroscopic evaluation,” Int. J. Pharmaceutics    132:107-113.-   Hovgaard et al., (1996) “Stabilization of insulin by    alkylmaltosides. B. Oral absorption in vivo in rats,” Int. J.    Pharmaceutics 132:115-121.-   Tetsuaki et al. (1997) “Lysis of Bacillus subtilis cells by glycerol    and sucrose esters of fatty acids,” Applied and Environmental    Microbiology, 53(3):505-508.-   Watanabe et al., (2000) “Antibacterial carbohydrate monoesters    suppressing cell growth of Streptococcus mutan in the presence of    sucrose,” Curr Microbiol 41(3): 210-213.

Although the present process has been described with reference tospecific details of certain embodiments thereof in the above examples,it will be understood that modifications and variations are encompassedwithin the spirit and scope of the invention. Accordingly, the inventionis limited only by the following claims.

What is claimed is:
 1. A method of increasing the bioavailability of an oligonucleotide in a subject by administering a composition comprising the oligonucleotide and an absorption increasing amount of an alkylglycoside to the subject via an oral, rectal or gastrointestinal delivery route, wherein the alkylglycoside comprises a maltose linked by glycosidic linkage to an alkyl chain comprising between 10 to 16 carbons, thereby increasing the bioavailability of the oligonucleotide in the subject.
 2. The method of claim 1, wherein the oligonucleotide is an RNA interference molecule.
 3. The method of claim 1, wherein the oligonucleotide is an antisense oligonucleotide.
 4. The method of claim 1, wherein the oligonucleotide contains one or more phosphorothioate linkages.
 5. The method of claim 1, wherein the alkylglycoside is selected from the group consisting of dodecyl maltoside, tridecyl maltoside, and tetradecyl maltoside.
 6. The method of claim 1, wherein the alkylglycoside is tetradecyl-beta-D-maltoside.
 7. The method of claim 1, wherein the oligonucleotide has a molecular weight from about 5-10 kD.
 8. The method of claim 1, wherein the alkylglycoside is dodecyl-beta-D-maltoside.
 9. The method of claim 1, wherein the composition further comprises citric acid, sodium citrate, propylene glycol, glycerin, ascorbic acid, sodium metabisulfite, ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride, sodium hydroxide, or combinations thereof.
 10. The method of claim 9, wherein the composition further comprises ethylenediaminetetraacetic acid (EDTA).
 11. The method of claim 1, wherein the composition is administered via the oral delivery route.
 12. The method of claim 1, wherein the composition is administered via the rectal delivery route.
 13. The method of claim 1, wherein the composition is administered via the gastrointestinal delivery route.
 14. The method of claim 1, wherein the alkylglycoside is present in the composition at a concentration of 0.01% to 20% by weight.
 15. The method of claim 1, wherein the oligonucleotide comprises a modified sugar moiety, a modified base moiety, a modified sugar linking moiety, or combination thereof.
 16. The method of claim 1, wherein the oligonucleotide comprises a modified linkage selected from the group consisting of phosphoramide, phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester, phosphoramidate, O-methylphosphoroamidite, peptide, and combinations thereof. 